Team:Freiburg Bioware/NoteBook/Labjournal/October2

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1 150. labday 15.10.2010
2 151. labday 16.10.2010
3 152. labday 17.10.2010
4 153. labday 18.10.2010
5 154. labday 19.10.2010
6 155. labday 20.10.2010
- 6.1 Midi-Prep
7 156. labday 21.10.2010
- 7.1 ÄKTA Chromatography and Ultrafiltration of virus particles
- 7.2 MTT Assay: Testing Superconstructs
8 157. labday 22.10.2010
- 8.1 SDS PAGE and Coomassie staining
9 158. labday 23.10.2010
10 159. labday 24.10.2010
- 10.1 Midi-Prep
11 160. labday 25.10.2010
12 161. labday 26.10.2010
13 162. labday 27.10.2010
14 163. labday 28.10.2010
15 164. labday 29.10.2010
16 165. labday 30.10.2010
17 166. labday 31.10.2010

150. labday 15.10.2010

Cloning CFP (from P666: PSB1C3_CFP) into pSB1C3_leftITR_CMV_beta-globin (P729)

Investigator Patrick
Digestions, 2 h 10 minutes, 37 °C:

P666: 5 µl DNA, 2 µl BSA, 2 µl Buffer 4 (10x), 1 µl Xba, 1 µl PstI, 9 µl H2O
P729: 4 µl DNA, 2 µl BSA, 2 µl Buffer 4 (10x), 1 µl SpeI, 1 µl PstI, 10 µl H2O

Expected results for the 1% agarose gel:

P666: about 2100 and 750 bp
P729: about 3300 and 20 bp

The gelextraction ...

P728: 11,8 ng/µl
P822: 34,6 ng/µl

... and following ligation (2,5 µl Insert, 5,5 µl vector, 1 µl T4 DNA Ligase, 1 µl T4 DNA Ligase Buffer (10x), 40 minutes, RT) were performed according to the standard protocol. After the transformation (with XL1B) the cells were plated and put into the 37°C room. Two additional transformations were performed with ligations from Volker labeled: "ligation viral brick 453 empty" & "viral brick 587 empty".

The following day the plates were checked for clones. Unfortunately there grew no clones on these two plates contrary to my plate with a a lot of clones.

Midi-Prep

Investigator: Chris W.

Midi-Prep of:

pSB1C3_001_RC_IRCK_P5tataless clone 1 =P866 =B516
pSB1C3_001_CMV_VP123_587-KO_Z34C_spacer clone2 =P867 =B526
pSB1C3_001_CMV_VP123_587-KO_Z34C clone2 =P868 =B529
pSB1C3_CMV_Zegfr:1907_MiddleLinker_VP2/3_587-KO_BAP clone 1 =P869 =B680
pSB1C3_CMV_Zegfr:1907_MiddleLinker_VP2/3_587-KO_6xHis clone 1 =P870 =B200

The Midi-Preps were performed according to the standard protocol yielding the following concentrations:

plasmid-no.	P866	P867	P868	P869	P870
concentration (ng/µl)	899,80	954,46	406,97	1642,76	1585,12

mini prep of several constructs

Investigator: Kira

c(rep52_1)=299,04 ng/ul
c(rep52_2)=290,07 ng/ul
c(rep78_1)=142,32 ng/ul
c(rep78_2)=175,36 ng/ul

Cell culture

Investigator: Kira

The cells are still alive. Medium was exchanged.--> RNA will be harvested tomorrow

Mini-Prep and test digestion of pSB1C3_CD_SDM-PstI_hGH_rITR

Investigator: Stefan

Glycerol stocks were prepared:

B694 = pSB1C3_CD_SDM-PstI_hGH_rITR clone 1
B695 = pSB1C3_CD_SDM-PstI_hGH_rITR clone 2

Mini-Prep was performed according to standard protocol:

P875 = pSB1C3_CD_SDM-PstI_hGH_rITR clone 1 c = 73,1 ng/µl
P876 = pSB1C3_CD_SDM-PstI_hGH_rITR clone 2 c = 78,3 ng/µl

Test digestion:

Components	P875 + P876 / µl
DNA	4
Buffer 4	1
BSA (10x)	1
XbaI	0,3
AgeI	0,3
H₂O	3,4
Total volume	10

Gel:
0,5g agarose, 50 ml TAE (1%), 3 µl GELRED, 115 Volt, running time ~50 minutes

Comment: Test digestion looks allright, cloning will be continued using P876.

151. labday 16.10.2010

Biobrick assembly: pSB1C3_lITR_CMV_ß-globin_CD_hGH_rITR and pSB1C3_lITR_phTERT_ß-globin_CD_hGH_rITR

Investigator: Achim

Plasmids:

P729: pSB1C3_lITR_CMV_ß-Globin
- c= 243.4 ng/µl
P730: pSB1C3_lITR_pHTERT_ß-Globin
- c= 81.1 ng/µl
P876: pSB1C3_CD_SDM-PstI_hGH_rITR
- c= 78.3 ng/µl

Digestion:

components	I1 (P729)	I2 (P730)	V (P876)
DNA	6	14	14
BSA (10x)	2	2	2
Buffer 4 (10x)	2	2	2
Enzyme EcoI	1	1	1
Enzyme XbaI	-	-	1
Enzyme SpeI	1	1	-
H2O	8	-	-
Total	20	20	20

Digestion: 2h, 37°C

Prep. gel:

0,8%, run for 45 min

Expected Bands: I1: 1335, I2: 1138, V: 4000

Corresponding bands were cut out

Gel ex.

Nanodrop concentrations:
- I1: 37.54 ng/µl
- I2: 25.38 ng/µl
- V: 26.47 ng/µl

Ligation:

ligation name	I1 + V	I2 + V
volume of vector	4.69	4.23
volume of insert	3.31	3.77
T4 ligase buffer (10x)	1	1
T4 ligase	1	1

Ligation @ RT for 40 min

Trafo:

Done by Kira

Mini-prep of mutual pSB1C3_lITR_phTERT_betaglobin_mGMK_TK30_hGH_rITR_SDM-PstI

Investigator Patrick

Yielded concentrations & given numbers:

pSB1C3_lITR_phTERT_betaglobin_mGMK_TK30_hGH_rITR_SDM-PstI clone 1: 208,4 ng/µl , P877 / B696
pSB1C3_lITR_phTERT_betaglobin_mGMK_TK30_hGH_rITR_SDM-PstI clone 2: 251,6 ng/µl , P878 / B696
pSB1C3_lITR_phTERT_betaglobin_mGMK_TK30_hGH_rITR_SDM-PstI clone 3: 212,6 ng/µl , P889 / B696

Test-digestion: 0,5 µl SpeI, 0,5 µl PstI, 3 µl DNA, 1 µl Buffer 4, 1 µl BSA, 4 µl H2O, 40 minutes, 37°C
Expected results: fragments with about 650 and 4900 bp

Obviously, this test digestion has to be repeated.

Preparations for tomorrow:

Mini-prep of 3 mutual pSB1C3_leftITR_CMV_beta-globin_CFP clones (have to be picked from the plate)
Midi-prep of pHelper
Midi-prep of B689:pSB1C3_lITR_CMV_betaglobin_mGMK_TK30_SDM-PstI_hGH_rITR clone 2

Biobrick assembly of Rep78 and Rep52

Investigator: Kira
Comment: After replacing the mutated Rep parts by the ordered Rep parts, PCR amplification has to be done in order to produce a biobrick. PCR program:

c(Rep52)=299 ng/ul
c(Rep78)=175 ng/ul

Rep52: praefix 094 & suffix 097
Rep78: praefix 093 & suffix 097

components	volume in µl
5x Phusion HF buffer	10
10 mM dNTP mix	1
primer_for (1:10 dilution)	2,5
primer_rev (1:10 dilution)	2,5
DNA template (1:100)	0,5
DMSO	0,5
Phusion polymerase	0,5
H₂O	32,5
Total volume (e.g. 50 µl)	50

Cycles	Temperature	Time
	98°C	30 sec
10x	98°C	15 sec
	63°C	25 sec
	72°C	32 sec
20x	98°C	15 sec
	66°C	25 sec
	72°C	32 sec
1x	72°C	5 min
Hold 4°C

1% agarose gel

Digestion of plasmid backbone:

pSB1C3_001 is used as backbone

Components	<b>vector Volume/µL
DNA	3,5 µl
BSA (10x)	2 µl
Buffer no. 4 (10x)	2,0 µl
Enzyme 1 EcoRI-HF	0,5 µl
Enzyme 2 SpeI	1,0 µl
H₂O	15 µl
Total volume	25

incubation @ 37 C for approx. 2 h

1% agarose gel

Digestion of PCR product:

Components	PCR product Volume/µL
DNA	35,0 µl
BSA (100x)	0,45 µl
Buffer no. 4	4,5 µl
Enzyme 1 EcoRI-HF	1,5 µl
Enzyme 2 SpeI	2,0 µl
H₂O	1,5 µl
Total volume	45

incubation @ 37 C for approx. 2 h

T4 ligation for 40 min
Transformation according to the standard protocol

RNA harvesting

Investigator: Kira
After transfection, the cells were incubated for 48 hours. Today, the cells will be harvested and RNA extracted, in order to perform RT-PCR and an additional PCR for evaluation of promoter activity.

The transfected cells were trypsinised and centrifuged for 2 min. The supernatant was discarded and pellet washed 2x with PBS. RNeasy Kit [Qiagen] was used for RNA extraction according to the manufacturer protocol.

c(CMV)= 335,69 ng/ul
c(P40)= 857,92 ng/ul
c(AAV_RC)= 760,21 ng/ul

152. labday 17.10.2010

Test digestion of pSB1C3_lITR_CMV_ß-globin_CFP

Investigator: Anna

Vector name:
pSB1C3_lITR_CMV_betaglobin_CFP_cl1 (P880): c = 452,67 ng/µl
pSB1C3_lITR_CMV_betaglobin_CFP_cl2 (P881): c = 288,88 ng/µl
pSB1C3_lITR_CMV_betaglobin_CFP_cl3 (P882): c = 288,36 ng/µl

Test Digestion:

components	volume P880 - P882 /µl	volume P434 /µl
DNA	2	2
BSA (10x)	1	1
Buffer 4 (10x)	1	1
Enzyme NgoMIV	0,3	0,3
Enzyme AgeI	0,3	0,3
H2O	5,4	5,4
Total volume	10	10

Gel:
0,5 g Agarose, 50 ml TAE (1%), 3 µl GELRED , at 115 Volt

Freiburg10 Test digestion of pSB1C3 lITR CMV ß-globin CFP.jpg

Cloning of hGH_rITR into pSB1C3_lITR_CMV_betaglobin_CFP

Investigator: Stefan

Cloning of our last GOI!

Vector name:
pSB1C3_lITR_CMV_betaglobin_CFP cl 1-3 (P880-P882)

Insert name:
pSB1C3_hGH_rITR (P728)

Digestion:

components	volume P880 - P882 /µl	volume P728 /µl
DNA	3	8
BSA (10x)	2	2
Buffer 4 (10x)	2	2
Enzyme PstI	1	1
Enzyme XbaI	-	1
Enzyme SpeI	1	-
H2O	11	4
Total volume (e.g. 15,20,25,30 µl)	20	20

Gel:
0,5 g Agarose, 50 ml TAE (1%), 3 µl GELRED , at 115 Volt

Test digestion of all constructs looked alright, therefore, cloning was continued using P881 only.

Gel extraction:
Was performed according to protocol.

T4 Ligation:

ligation name	728 + 881
volume of vector	2,68
volume of insert	5,32
T4 ligase buffer (10x)	1
T4 ligase	1

Transformation:
Transformation was performed according to standard protocol using BL21 cells.

RT-PCR

Investigator: Kira
For further experiments, RNA has to be translated into cDNA. The PCR was performed according to the manufacturer protocol.

153. labday 18.10.2010

quantitative real-time PCR for detection of virus titer

Investigator: Achim

qPCR of harvested virus particles to determine the virus titers of our different constructs

Total number of samples: 58

Test-digestion of pSB1C3_lITR_phTERT_betaglobin_mGMK_TK30_hGH_rITR_SDM-PstI clone 1, 2 and 3 (P877, 878 and 879)

Investigator Patrick

Check the plasmid for leftITR: 0,5 µl EcoRI, 1 µl BstEII, 7 µl DNA, 2 µl Buffer 4, 2 µl BSA, 7,5 µl H2O, 45 minutes 37°C, 45 minutes 60°C

Check the plasmid for hGH_rITR and ... : 0,5 µl AgeI, 0,5 µl PstI, 3 µl DNA, 1 µl BSA, 1 µl Buffer 4, 4 µl H2O, 70 minutes 37°C

Expected results:

leftITR: about 190 bp
hGH_rITR: about 670 bp

Unfortunately the digestions had to be reapeated because i didnt switch on the current so the samples and especially the 1kb GeneRuler marker diffused.

The second run: see above

PstI or BstEII seems to work not properly

MTT Assay

Investigator Kerstin, Anissa

Results:

Freiburg10 Results of MTTAssay 18 10 10 2day 3pic.jpg

Freiburg10 Results of MTTAssay 18 10 10 2day 2pic.jpg

Mini-Prep and test digestion of several constructs

Investigator: Jessica

Glycerol stocks were prepared:

B702 = pSB1C3_lITR_pTERT_ßglobin_CD_hGH_rITR clone 1
B703 = pSB1C3_lITR_pTERT_ßglobin_CD_hGH_rITR clone 2
B704 = pSB1C3_001_VP3 clone 1
B705 = pSB1C3_001_VP3 clone 2
B706 = pSB1C3_001_VP2 clone 1
B707 = pSB1C3_001_VP2 clone 2
B708 = pSB1C3_001_Rep78 clone 1
B709 = pSB1C3_001_Rep78 clone 2
B710 = pSB1C3_001_Rep52 clone 1
B711 = pSB1C3_001_Rep52 clone 2
B712 = pSB1C3_lITR_CMV_ßglobin_CD_hGH_rITR clone 1
B713 = pSB1C3_lITR_CMV_ßglobin_CD_hGH_rITR clone 2
B714 = pSB1C3_001_VP1 clone 1
B715 = pSB1C3_001_VP1 clone 2

Mini-Prep was performed according to standard protocol:

P886 = pSB1C3_lITR_pTERT_ßglobin_CD_hGH_rITR clone 1 c= 232,2ng/µl
P887 = pSB1C3_lITR_pTERT_ßglobin_CD_hGH_rITR clone 2 c= 186,2ng/µl
P888 = pSB1C3_001_VP3 clone 1 c= 300,8ng/µl
P889 = pSB1C3_001_VP3 clone 2 c= 284,4ng/µl
P890 = pSB1C3_001_VP2 clone 1 c= 298,3ng/µl
P891 = pSB1C3_001_VP2 clone 2 c= 299,9ng/µl
P892 = pSB1C3_001_Rep78 clone 1 c= 143,9ng/µl
P893 = pSB1C3_001_Rep78 clone 2 c= 163,4ng/µl
P894 = pSB1C3_001_Rep52 clone 1 c= 166,6ng/µl
P895 = pSB1C3_001_Rep52 clone 2 c= 181,6ng/µl
P896 = pSB1C3_lITR_CMV_ßglobin_CD_hGH_rITR clone 1 c= 250,5ng/µl
P897 = pSB1C3_lITR_CMV_ßglobin_CD_hGH_rITR clone 2 c= 173,4ng/µl
P898 = pSB1C3_001_VP1 clone 1 c= 272,7ng/µl
P899 = pSB1C3_001_VP1 clone 2 c= 294,8ng/µl
P900 = pSB1C3_hGH_rITR (from B160) c= 136,7ng/µl

Test digestion:

Components	P886,887,892,893,894,895,896,897 / µl	P888,889,890,891898,899 / µl
DNA	1,5	1,5
Buffer	(4) 1	(2) 1
BSA (10x)	1	1
NgoMIV	0,4	-
XbaI	0,4	-
PstI	-	0,6
XcmI	-	0,4
H₂O	4,5	4,5
Total volume	10	10

Gel:
1,0g agarose, 100 ml TAE (1%), 6 µl GELRED, Volt, running time minutes

Comment: Rep 52/78 will be checked,pSB1C3_lITR_CMV_ßglobin_CD_hGH_rITR and pSB1C3_lITR_pTERT_ßglobin_CD_hGH_rITR will be sequenced

PCR of mGMK and SR39

Investigator: Anna

Plasmids:
pSB1C3_mGMK_TK30_SDM-PstI clone 2(P804)
pSB1C3_mGMK_sr39 clone 1(P860)

Oligos:
O193: pTK30_for
O81: pmgmk_tk30_suffix_RFC25_rev

PCR Mix:

Components	Volume /µl
Phusion Buffer	10
dNTP	1
Primer_for	2,5
Primer_rev	2,5
DNA template	1
H2O	32,5
Total volume	50

PCR Program:

Cycles	Temperature	Time
	98°C	60 sec
	98°C	15 sec
8x	52°C	25 sec
	72°C	25 sec
	98°C	15 sec
17x	67°C	25 sec
	72°C	25 sec
1x	72°C	5 min
Hold 4°C

Gel:
0,5 g Agarose, 50 ml TAE (1%), 3 µl GELRED , at 115 Volt

[[Image:|550px|]]

154. labday 19.10.2010

Midi-Prep

Investigator: Chris W.

Midi-Prep of:

pSB1C3_lITR_CMV_betaglobin_mVenus_hGH_rITR clone1 =P901 =B200
pSB1C3_lITR_phTERT_betaglobin_mGMK_TK30_hGH_rITR_SDM-PstI clone 2 =P902 =B697

The Midi-Preps were performed according to the standard protocol yielding the following concentrations:

plasmid-no.	P901	P902
concentration (ng/µl)	1563,63	1348,26

Continuation of PCR of mGMK and SR39

Investigator: Jessica

vector (P320) and PCR product was digested

Components	P320 / µl	PCR product P804 and P860 / µl
DNA	1,5	20
Buffer	2	3
BSA (10x)	2	3
AgeI	1	1,5
XbaI	1	1,5
H₂O	14,5	1
Total volume	20	30

Ligation

P320 c= 5,08 ng/µl
P804 c= 11,73 ng/µl
P860 c= 26,57 ng/µl

P320 + P804: 4,93µl : 3,07µl
P320 + P860: 6,28µl : 1,72µl

Transformation with BL21 and Cm

Cloning of CMV into pSB1C3_001_VP1, pSB1C3_001_VP2 and pSB1C3_001_VP3

Investigator: Kerstin, Anna

Plasmids:

P888: pSB1C3_001_VP3, c = 300,8 ng/µl
P890: pSB1C3_001_VP2, c = 298,3 ng/µl
P898: pSB1C3_001_VP1, c = 272,7 ng/µl
P727: pSB1C3_001_CMV, c = 225,5 ng/µl

Digestion:

components	VP3	VP2	VP1	CMV
DNA	4	4	4	8
BSA (10x)	2	2	2	2
Buffer 4 (10x)	2	2	2	2
Enzyme EcoI	1	1	1	1
Enzyme XbaI	1	1	1	-
Enzyme SpeI	-	-	-	1
H2O	10	10	10	10
Total	20	20	20

Digestion: 2h @ 37°C

Gel:

1% agarose gel, 1 µl Gelred, run for 45 min

Gel extraction

sample name	VP3	VP2	VP1	CMV
nanodrop concentrations	44,36	32,33	22,73	18,5
expected fragment size	4100	4000	3700	650

Ligation:

ligation name	VP3 + CMV	VP2 + CMV	VP1 + CMV
volume of vector	5,7	4,3	5
volume of insert	3,3	3,7	3
T4 ligase buffer (10x)	1	1	1
T4 ligase	1	1	1

Ligation @ RT for 30 min

Trafo:

Was done following the standard protocol using BL21 cells.

155. labday 20.10.2010

Midi-Prep

Investigator: Chris W.

Midi-Prep of:

pSB1C3_lITR_pTERT_ßglobin_CD_hGH_rITR clone 1 =P903 =B702
pSB1C3_lITR_CMV_ßglobin_CD_hGH_rITR clone 1 =P904 =B712

The Midi-Preps were performed according to the standard protocol yielding the following concentrations:

plasmid-no.	P903	P904
concentration (ng/µl)	832,63	1174,49

156. labday 21.10.2010

ÄKTA Chromatography and Ultrafiltration of virus particles

Investigator: Hanna
ÄKTA chromatography with VP1up_NLS_mVenus_VP2/3 containing virus particles was conducted. Fraction 5 - 10 delivered highest protein concentrations.

Sample	A(260 nm)	A(280 nm)	A(515 nm) (YFP)
5	0.032	0.027	0.003
6	0.019	0.019	0.003
7	0.075	0.09	0.01
8	0.054	0.075	0.007
10	-0.008	-0.008	0.005

A further attempt was conducted which included digestion with Benzonase (1 hour) prior to ÄKTA chromatography. Following protein concentrations were obtained:

Sample	A(260 nm)	A(280 nm)	A(515 nm) (YFP)
5	0.005	0.007	0.003
6	0.047	0.041	0.006
7	0.151	0.153	0.01
8	0.172	0.2	0.009
9	0.098	0.128	0.009
10	0.053	0.074	0.005

Ultrafiltration
Ultrafiltration of CFP_MiddleLinker_VP2/3 containing virus particles and 587-BAP virus particles were concentrated via Vivaspin-Ultrafiltration:

20 mL virus containing cell culture supernatant was added to GE Vivaspin 20 filter and centrifuged with 4000 g at 15°C until 750 - 1000 µL was left-
5 mL Bis-Trus buffer (pH 6) was added and centrifuged again with 4000 g at 15°C (washing).
This step was repeated 3 more times.
Membrane was carefully resuspended and cleared. Suspension was transfered to low-binding eppi and centrifuged with 10000 g for 10 minutes at 15°C.
Supernatant was transfered to new low-binding eppi and again centrifuged with 10000 g for 10 minutes at 15°C.
Supernatant was transfered to new low-binding eppi and stored at 4°C over night. To do: ÄKTA chromatography.

MTT Assay: Testing Superconstructs

Investigator: Anissa, Kerstin

157. labday 22.10.2010

SDS PAGE and Coomassie staining

Investigator: Hanna

Prior to performing Western Blot we decided to investigate running behaviour of different samples.

1. Cell debris (control)
2. Cell debris containing virus particles
3. Concentrated virus stock (containing CFP_MiddleLinker_VP2/3)
4. ÄKTA purified virus stock: Fraction 6
5. ÄKTA purified virus stock: Fraction 7
6. Benzonase treated, ÄKTA purified virus stock: Fraction 7
7. Benzonase treated, ÄKTA purified virus stock: Fraction 8

5 µL Laemmli buffer was added to 20 µL sample. Samples were incubated at 95°C for 8 minutes and loaded onto a SDS gel (10 %). SDS PAGE was performed at 90 V (collection gel) resp. 120 V (separation gel).
Gel was put into Coomassie dye, heated for 30 seconds in microwave and incubated for 1 hours shaking.
Gel was decolorized in acetic acid (20%).
Loading plan:

Marker

Concentrated Stock

ÄKTA 6

ÄKTA 7

Benzonase/ÄKTA 7

Benzonase/ÄKTA 8

- - -

Cell debris

Cell debris + Virus

Gel picture shows that concentration works :)
In addition to that one can see that the BSA bands disappear after ÄKTA chromatography.
Next step: Western Blot of ÄKTA purified virus stocks.

158. labday 23.10.2010

159. labday 24.10.2010

Midi-Prep

Investigator: Chris W.

Midi-Prep of:

pSB1C3_001_RC_IRCK_VP2-ko_HSPG-ko_P5tataless cl1 =P966 =B523

The Midi-Prep were performed according to the standard protocol yielding the following concentration:

plasmid-no.	P966
concentration (ng/µl)	310,69

160. labday 25.10.2010

161. labday 26.10.2010

162. labday 27.10.2010

163. labday 28.10.2010

164. labday 29.10.2010

165. labday 30.10.2010

166. labday 31.10.2010

=> Go to Labjournal November (labday 167 - 170 )

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 <li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/September">September part 1 (labday 107 - 123)</a></li>
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-<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October3">October part 3 (labday 156 - 166 )</a></li>
 <li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/November">November  (labday 167 - 170 )</a></li>
-<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/Cellculture">Cellculture </a></li>
+<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/Cellculture">Cellculture</a></li>
 </ul>
 </div>
@@ Line 35: / Line 35: @@
 <!-- NoteBook -->
-===<p style="font-size:17px; background-color:#00dd77;">146. labday 11.10.2010</p>===
+===<p style="font-size:17px; background-color:#00dd77;">150. labday 15.10.2010</p>===
-====<p style="font-size:15px; background-color:#66bbff;"><b>Colony PCR of Cloning VP2 Fusion and Super constructs into pSB1C3</b></p>====
+====<p style="font-size:15px; background-color:#66bbff;">Cloning CFP (from P666: PSB1C3_CFP) into pSB1C3_leftITR_CMV_beta-globin (P729)</p>====
-<b>Investigator: Achim, Hanna</b><br>
+'''Investigator Patrick <br>
-<br/>
+Digestions, 2 h 10 minutes, 37 °C:
-<b>Comment:</b> Because yesterday's cloning didn't deliver a good separartion of the expected gel bands. Nevertheless ligation and trafo was performed. In order to immediately find out, whether we received successful results a colony PCR will be performed. <br/>
+*P666: 5 µl DNA, 2 µl BSA, 2 µl Buffer 4 (10x), 1 µl Xba, 1 µl PstI, 9 µl H2O
-Two clones were picked from each plat. In addition to that a positive (pAAV_RC) and a negative control (pSB1C3_lITR) was prepared. <br/>
+*P729: 4 µl DNA, 2 µl BSA, 2 µl Buffer 4 (10x), 1 µl SpeI, 1 µl PstI, 10 µl H2O
-Used primer: 4200 rev and Cap3500 for. Expected fragment size: 885 bp. <br/>
-PCR was performed following the standard protocol. <br/>
-[[Image:Freiburg10 1110 testdigestionhanna1label.png|thumb|none|800px]]
+Expected results for the 1% agarose gel:
+*P666: about 2100 and 750 bp
+*P729: about 3300 and 20 bp
-[[Image:Freiburg10 1110 testdigestionhanna2label.png|thumb|none|800px]]
+<br>
+[[Image:Freiburg10_1015pat_fertig.jpg|thumb|center|600px]]
+<br>
+<br>
+The gelextraction ...
+P728: 11,8 ng/µl
+<br>
+P822: 34,6 ng/µl
+... and following ligation (2,5 µl Insert, 5,5 µl vector, 1 µl T4 DNA Ligase, 1 µl T4 DNA Ligase Buffer (10x), 40 minutes, RT) were performed according to the standard protocol. After the transformation (with XL1B) the cells were plated and put into the 37°C room.
+Two additional transformations were performed with ligations from Volker labeled: "ligation viral brick 453 empty" & "viral brick 587 empty". <br>
+The following day the plates were checked for clones. Unfortunately there grew no clones on these two plates contrary to my plate with a a lot of clones.
+====<p style="font-size:15px; background-color:#66bbff;">Midi-Prep</p>====
+'''Investigator: Chris W. <br>'''
+<p style="font-size:13px; color:#003399;"> Midi-Prep of:</p><br/>
+pSB1C3_001_RC_IRCK_P5tataless clone 1 =P866 =B516<br/>
+pSB1C3_001_CMV_VP123_587-KO_Z34C_spacer clone2 =P867 =B526<br/>
+pSB1C3_001_CMV_VP123_587-KO_Z34C clone2 =P868 =B529<br/>
+pSB1C3_CMV_Zegfr:1907_MiddleLinker_VP2/3_587-KO_BAP clone 1 =P869 =B680<br/>
+pSB1C3_CMV_Zegfr:1907_MiddleLinker_VP2/3_587-KO_6xHis clone 1 =P870 =B200<br/>
-All samples match the positive control!
-<br/>
-<b>To do:</b> Mini-Prep and sequencing.
-<br/>
-====<p style="font-size:15px; background-color:#66bbff;"><b>Preparation of SDS-PAGE gel (10%)</b></p>====
-<b>Investigator: Hanna</b><br>
-<br/>
-<b>Comment:</b> In order to perform a Western Blot of different virus capsids (with and without capsid-motifs), 2 10% SDS polyacrylamid gels were prepared. <br/>
-<br/>
-<b>Resolving gel: 15 mL</b>
-<br/>
-* H<sub>2</sub>O: 5.9 mL
-* Acryl-bisacrylamide mix (30%): 5 mL
-* Tris (1.5 M, pH 8.8): 3.8 mL
-* SDS (10%): 0.15 mL
-* Ammonium persulfate (10%): 0.15 mL
-* TEMED: 0.006 mL
 <br/>
+The Midi-Preps were performed according to the standard protocol yielding the following concentrations:
-<b>Stacking gel (5%): 5 mL </b>
+{| border="1"
+| plasmid-no. || align="right" |P866|| align="right" |P867|| align="right" |P868|| align="right" |P869|| align="right" |P870
+|-
+| concentration (ng/µl)|| align="right" |899,80 || align="right" |954,46 || align="right" |406,97|| align="right" |1642,76|| align="right" |1585,12
+|}
+<br>
 <br/>
-* H<sub>2</sub>O: 3.4 mL
+====<p style="font-size:15px; background-color:#66bbff;">mini prep of several constructs</p>====
-* Acryl-bisacrylamide mix (30%): 0.83 mL
+'''Investigator:  Kira <br/>
-* Tris (1.5 M, pH 6.8): 0.63 mL
-* SDS (10%): 0.05 mL
+c(rep52_1)=299,04 ng/ul<br/>
-* Ammonium persulfate (10%): 0.05 mL
+c(rep52_2)=290,07 ng/ul<br/>
-* TEMED: 0.005 mL
+c(rep78_1)=142,32 ng/ul<br/>
+c(rep78_2)=175,36 ng/ul<br/>
+====<p style="font-size:15px; background-color:#66bbff;">Cell culture</p>====
+'''Investigator:  Kira <br/>
+The cells are still alive. Medium was exchanged.--> RNA will be harvested tomorrow
+====<p style="font-size:15px; background-color:#66bbFF;"><b>Mini-Prep and test digestion of pSB1C3_CD_SDM-PstI_hGH_rITR</b></p>====
+<b>Investigator: Stefan</b><br />
+Glycerol stocks were prepared:<br>
-====<p style="font-size:15px; background-color:#66bbff;"><b>Colony PCR of p40_VP123_capins</b></p>====
-<b>Investigator: Bea</b><br>
-<p style="color:#66bbff;"><i>Comment</i>: Since the first attempt did not work,and no cells grew on the plate and the same ligation was transformed again into BL-21 and a lot of clones grew on the plate, I decided to perform a colony PCR in order to check several colonies and to inoculate at the same day for a Midi-Prep. </p>
-<b>Protocol:</b><br />
 <ul>
-<li>Primer used: O162</li>
+<li>B694 = pSB1C3_CD_SDM-PstI_hGH_rITR clone 1</li>
-<li>Primer used: O38</li>
+<li>B695 = pSB1C3_CD_SDM-PstI_hGH_rITR clone 2</li>
+</ul><br>
+Mini-Prep was performed according to standard protocol:<br>
+<ul>
+<li>P875 = pSB1C3_CD_SDM-PstI_hGH_rITR clone 1 c = 73,1 ng/µl</li>
+<li>P876 = pSB1C3_CD_SDM-PstI_hGH_rITR clone 2 c = 78,3 ng/µl</li>
 </ul>
-[[Image: Freiburg10_ColonyPCR_p40vp123_1110.PNG|thumb|center|600px]]
-<br />
-The PCR products were loaded on a 1% agarose gel. The results can be seen above in the gel picture: <br />
-<br />
-<b>Result: We can see two things: The cloning of p40 to VP123 worked quiet well AND the Robust PCR Kit which was used for the first time worked as well.</b>
-<br />
-[[Image: Freiburg10_ColonyPCR_p40VP123.png|thumb|center|400px]]
-<br />
-====<p style="font-size:15px; background-color:#66bbff;"><b>Cloning of lITR_CMV_betaglobin and lITR_phTERT_betaglobin into pSB1C3_CD</b></p>====
+Test digestion:<br>
-<b>Investigator: Stefan</b><br>
+{| border="1"
+| align="left" | '''Components''' ||align="left"| <b>P875 + P876 / µl</b>
+|-
+| align="left" | DNA   ||align="left"| 4
+|-
+| align="left" | Buffer 4 ||align="left"| 1
+|-
+| align="left" | BSA (10x)  ||align="left"| 1
+|-
+| align="left" | XbaI ||align="left"| 0,3
+|-
+| align="left" | AgeI ||align="left"| 0,3
+|-
+| align="left" | H<sub>2</sub>O ||align="left"| 3,4
+|-
+| align="left" | '''Total volume''' ||align="left"| <b>10</b>
+|}
-<p style="font-size:13px; color:red;">Comment: To produce another GOI for testing in cell culture, the cytosine deaminase needs to be assembled with lITR_promotor_betaglobin. In the next step hgH_rITR needs to be added.</p><br />
+Gel:<br>
+,5g agarose, 50 ml TAE (1%), 3 µl GELRED, 115 Volt, running time ~50 minutes<br>
-Vector name:<br />
+[[Image:Freiburg10 td151010.jpg|550px|thumb|center]]
-pSB1C3_CD clone 1 <br />
+<p style="color:#66bbff;"><i>Comment</i>: Test digestion looks allright, cloning will be continued using P876.</p>
-pSB1C3_CD clone 2 <br />
-Insert name:<br />
+===<p style="font-size:17px; background-color:#00dd77;">151. labday 16.10.2010</p>===
-pSB1C3_lITR_CMV_beta-globin (P729)<br />
+====<p style="font-size:15px; background-color:#66bbff;">Biobrick assembly: pSB1C3_lITR_CMV_ß-globin_CD_hGH_rITR and pSB1C3_lITR_phTERT_ß-globin_CD_hGH_rITR </p>====
-pSB1C3_lITR_phTERT_beta-globin (P730)<br />
-<b>Digestion:</b><br /><br />
+'''Investigator: Achim'''
+'''Plasmids:'''
+*P729: pSB1C3_lITR_CMV_ß-Globin
+**c= 243.4 ng/µl
+*P730: pSB1C3_lITR_pHTERT_ß-Globin
+**c= 81.1 ng/µl
+*P876: pSB1C3_CD_SDM-PstI_hGH_rITR
+**c= 78.3 ng/µl
+'''Digestion:'''
-<br />
 {| border="1"
-| <b>components</b>   || align="right" |<b>volume CD clone 1 + 2  /µl</b> || align="right" |<b>volume P729  /µl </b>||<b>volume P730  /µl</b>
+| <b>components</b>   || align="right" |<b>I1 (P729)</b> || align="right" |<b>I2 (P730)</b> || align="right" |<b>V (P876)
 |-
-| DNA  ||  align="right" |6 ||  align="right" |14||  align="right" |6
+| DNA  ||  align="right" |6 ||  align="right" |14||  align="right" |14
 |-
 | BSA (10x) ||  align="right" |2 ||  align="right" |2||  align="right" |2
 |-
-| Buffer 4 (10x)||  align="right" |2 ||  align="right" |2||  align="right" |2
+| Buffer 4 (10x)||  align="right" |2 ||  align="right" |2 ||  align="right" |2
 |-
 |Enzyme EcoI||  align="right" |1||  align="right" |1 ||  align="right" |1
 |-
-|Enzyme XbaI||  align="right" |1||  align="right" |- ||  align="right" |-
+|Enzyme XbaI||  align="right" |-||  align="right" |- ||  align="right" |1
 |-
-|Enzyme SpeI||  align="right" |-||  align="right" |1||  align="right" |1
+|Enzyme SpeI||  align="right" |1||  align="right" |1 ||  align="right" |-
 |-
-|H2O||  align="right" |8||  align="right" |- ||  align="right" |8
+|H2O||  align="right" |8||  align="right" |- ||  align="right" |-
 |-
-|'''Total volume (e.g. 15,20,25,30 µl)'''||  align="right" | 20||  align="right" | 20||  align="right" | 20
+|'''Total '''||  align="right" | 20||  align="right" | 20||  align="right" | 20
 |}
-<br />
+Digestion: 2h, 37°C
-<b>Gel:</b><br />
-,5 g Agarose, 50 ml TAE (1%), 3 µl GELRED , at 115 Volt<br />
+'''Prep. gel:'''
+*0,8%, run for 45 min
-<br/>
+[[Image:Freiburg10 1610 bba.png|thumb|none|400px|Expected Bands: I1: 1335, I2: 1138, V: 4000]]
-[[Image:Freiburg10 cloning 111010.jpg|550px|]]<br/>
-<br/>
+*Corresponding bands were cut out
-<p style="font-size:13px; color:red;"> CD clone 1 yielded to bands around 2500 bp to 3000 bp. Since the vector was cut only using EcoRI and SpeI, it was expected to be linearized, not to be cut into two fragments this size. Therefore, this sample was discarded and cloning was continued using CD clone 2. </p><br />
+'''Gel ex.'''
+*Nanodrop concentrations:
+**I1: 37.54 ng/µl
+**I2: 25.38 ng/µl
+**V: 26.47 ng/µl
-<b>Gel extraction</b>: <br>
+'''Ligation:'''
-Was performed according to protocol.
-<br>
-<b>T4 Ligation</b>: <br>
 {| border="1"
-|ligation name || align="right" |729 + CD cl2|| align="right" |730 + CD cl2
+|ligation name || align="right" |I1 + V|| align="right" |I2 + V
 |-
-|volume of vector || align="right" |3,67|| align="right" |2,82
+|volume of vector || align="right" |4.69|| align="right" |4.23
 |-
-|volume of insert|| align="right" |4,33|| align="right" |5,18
+|volume of insert|| align="right" |3.31|| align="right" |3.77
 |-
 |T4 ligase buffer (10x)|| align="right" |1|| align="right" |1
@@ Line 163: / Line 204: @@
 |}
-<br>
+*Ligation @ RT for 40 min
-<b>Transformation</b>: <br>
-Was performed according to standard protocol using BL21 cells.
-<br/>
-====<p style="font-size:15px; background-color:#66bbff;"><b>Seeding HT1080 and A431 for testing different concentrations of ganciclovir by MTT-Assay</b></p>====
+'''Trafo:'''
-<b>Investigator: Kerstin, Anissa</b><br>
+*Done by Kira
-*Seeding 4x 96-well plates: 2x HT1080 and 2x A431
-====<p style="font-size:15px; background-color:#66bbff;"><b>FACS-Analysis</b></p>====
+<br>
-<b>Investigator: Kerstin</b><br>
-...
+====<p style="font-size:15px; background-color:#66bbff;">Mini-prep of mutual pSB1C3_lITR_phTERT_betaglobin_mGMK_TK30_hGH_rITR_SDM-PstI</p>====
-====<p style="font-size:15px; background-color:#66bbff;"><b>Preparation of the ELISA</b></p>====
+'''Investigator Patrick
-<b>Investigator: Volker</b><br>
+<br>
+Yielded concentrations & given numbers:
+*pSB1C3_lITR_phTERT_betaglobin_mGMK_TK30_hGH_rITR_SDM-PstI clone 1:  208,4 ng/µl , P877 / B696
+*pSB1C3_lITR_phTERT_betaglobin_mGMK_TK30_hGH_rITR_SDM-PstI clone 2:  251,6 ng/µl , P878 / B696
+*pSB1C3_lITR_phTERT_betaglobin_mGMK_TK30_hGH_rITR_SDM-PstI clone 3:  212,6 ng/µl , P889 / B696
-The AAV particle standard that contains 3.6x10^9 viral particles was dissolved in 500µl as described in the protocol of the Progen AAV Titration ELISA and a absorption spectrum was measured.
-These purified and concentrated viral particles could be used for biophysical measurements, there for the possibility to detect the viral particles by absorption was interesting for us.
-The Spectrum measured in the NanoDrop is the following:
+<br>
-[[Image:Freiburg10_Absorption spectrum of the AAV particle standard.jpeg|thumb|center|800px]]
+Test-digestion: 0,5 µl SpeI, 0,5 µl PstI, 3 µl DNA, 1 µl Buffer 4, 1 µl BSA, 4 µl H2O, 40 minutes, 37°C
+<br>
+Expected results: fragments with about 650 and 4900 bp
+<br>
+[[Image:Freiburg10_1016pat_test.jpg‎|thumb|center|600px]]
+<br>
+Obviously, this test digestion has to be repeated.
-====<p style="font-size:15px; background-color:#66bbff;"><b>Trafo evaluation of Rep52</b></p>====
+Preparations for tomorrow:
-<b>Investigator:Kira</b><br>
+*Mini-prep of 3 mutual pSB1C3_leftITR_CMV_beta-globin_CFP clones (have to be picked from the plate)
-T4 ligation seems to worked out. The plate contained many colonies.
+*Midi-prep of pHelper
+*Midi-prep of B689:pSB1C3_lITR_CMV_betaglobin_mGMK_TK30_SDM-PstI_hGH_rITR clone 2
-===<p style="font-size:17px; background-color:#00dd77;">147. labday 12.10.2010</p>===
+====<p style="font-size:15px; background-color:#66bbff;">Biobrick assembly of Rep78 and Rep52</p>====
-====<p style="font-size:15px; background-color:#66bbFF;"><b>Sequencing analysis: pSB1C3_mGMK_TK30 and pSB1C3_CD</b></p>====
+'''Investigator: Kira <br />
-<b>Investigator: Stefan</b><br>
+'''Comment:''' After replacing the mutated Rep parts by the ordered Rep parts, PCR amplification has to be done in order to produce a biobrick.
+PCR program:
+<br />
-[[Image:Freiburg10 sequencingmGMK TK30 SDM PstI.jpg|550px]]<br>
+c(Rep52)=299 ng/ul <br />
-[[Image:Freiburg10 sequencing CD CFP.jpg|550px]]
+c(Rep78)=175 ng/ul <br />
+Rep52: praefix 094 & suffix 097 <br />
+Rep78: praefix 093 & suffix 097 <br />
-====<p style="font-size:15px; background-color:#66bbFF;"><b>Test digestion of pSB1C3_CD and pSB1C3_CD_SDM-PstI_new </b></p>====
-<b>Investigator: Stefan</b><br>
-<p style="color:#66bbff;"><i>Comment</i>: Nachricht von Stefan an Stefan: bitte ergänzen!</p>
+{| border="1"
+| components   || align="right" |volume in µl
+|-
+| 5x Phusion HF buffer  ||  align="right" | 10
+|-
+| 10 mM dNTP mix ||  align="right" |1
+|-
+|primer_for (1:10 dilution)||  align="right" |  2,5
+|-
+|primer_rev (1:10 dilution)||  align="right" | 2,5
+|-
+|DNA template (1:100)||  align="right" |  0,5
+|-
+|DMSO||  align="right" |  0,5
+|-
+|Phusion polymerase||  align="right" | 0,5
+|-
+|H<sub>2</sub>O||  align="right" | 32,5
+|-
+|'''Total volume (e.g. 50 µl)'''||  align="right" | 50
+|}
+<br />
+{| border="1"
+|Cycles||Temperature||Time
+|-
+|||98°C||30 sec
+|-
+|10x||98°C||15 sec
+|-
+|||63°C||25 sec
+|-
+|||72°C||32 sec
+|-
+|20x||98°C||15 sec
+|-
+|||66°C||25 sec
+|-
+|||72°C||32 sec
+|-
+|1x||72°C||5 min
+|-
+|Hold 4°C
+|}
+<br>
+% agarose gel <br />
+[[Image:Freiburg10_Rep78&Rep52_PCR.jpg]]
+Digestion of plasmid backbone:
-<p style="color:#66bbff;"><i>Comment</i>: Sequencing results revealed that there is CFP rather than CD in P819. In order to verify this and exclude the possibility that the wrong plasmid was sent for sequencing test digestions were performed. Additionally the original vector at which the SDM to remove the PstI was performed was digested as well to verify its insert.</p>
+pSB1C3_001 is used as backbone
 {| border="1"
-| align="left" | '''Components''' ||align="left"| <b>digest 1 / µl</b>||align="left"| <b>digest 2 / µl</b>
+| align="left" | '''Components''' ||align="left"| <b>vector</b> Volume/µL
 |-
-| align="left" | DNA   ||align="left"| 1||align="left"| 7
+| align="left" | DNA  ||align="left"| 3,5 µl
 |-
-| align="left" | Buffer 4 ||align="left"| 1||align="left"| 1
+| align="left" | BSA (10x) ||align="left"| 2 µl
 |-
-| align="left" | BSA (10x)  ||align="left"| 1||align="left"| 1
+| align="left" | Buffer no. 4 (10x) ||align="left"| 2,0 µl
 |-
-| align="left" | Enzyme I ||align="left"| SpeI 0,3 ||align="left"| PstI 0,3
+| align="left" | Enzyme 1 EcoRI-HF ||align="left"| 0,5 µl
 |-
-| align="left" | Enzyme II ||align="left"| XbaI 0,3 ||align="left"| XbaI 0,3
+| align="left" | Enzyme 2 SpeI  ||align="left"| 1,0 µl
 |-
-| align="left" | H<sub>2</sub>O ||align="left"| 6,4||align="left"| 0,4
+| align="left" | H<sub>2</sub>O ||align="left"| 15 µl
 |-
-| align="left" | '''Total volume''' ||align="left"| <b>10</b>  ||align="left"| <b>10</b>
+| align="left" | '''Total volume''' ||align="left"| <b>25</b>
 |}
+<br />
-<p style="color:#66bbff;"><i>Comment</i>: Using these digestion approaches, it was expected for CFP to deliver always bands at ~750 bp. For the CD it was expected to show bands at 90 bp, 1200 bp and 2000 bp if the CD still contains the PstI restictiton site, 1300 bp and 2000 bp if it was removed.</p>
+incubation @ 37 C for approx. 2 h
-Gel:<br>
+% agarose gel <br />
-,5g agarose, 50 ml TAE (1%), 3 µl GELRED, 115 Volt, running time ~50 minutes<br>
+[[Image:Freiburg10_digestion_pSB1C31_16.10.jpg|thumb|center|600px]]
-[[Image:Freiburg10 test digestion 121010 1.jpg|550px]]
+Digestion of PCR product:
+{| border="1"
+| align="left" | '''Components''' ||align="left"| <b>PCR product</b> Volume/µL
+|-
+| align="left" | DNA   ||align="left"| 35,0 µl
+|-
+| align="left" | BSA (100x) ||align="left"| 0,45 µl
+|-
+| align="left" | Buffer no. 4  ||align="left"| 4,5 µl
+|-
+| align="left" | Enzyme 1 EcoRI-HF ||align="left"| 1,5 µl
+|-
+| align="left" | Enzyme 2 SpeI  ||align="left"|2,0 µl
+|-
+| align="left" | H<sub>2</sub>O ||align="left"| 1,5 µl
+|-
+| align="left" | '''Total volume''' ||align="left"| <b>45</b>
+|}
+<br />
+incubation @ 37 C for approx. 2 h <br />
-<p style="color:#66bbff;"><i>Comment</i>: As it can be seen, P819 does contain CFP, therefore a mistakenly sequenced plasmid can be excluded. Also it can be seen that P690 contains the CD. Therefore an additonal SDM of P690 to remove the PstI restriction site is necessary.</p>
+T4 ligation for 40 min <br />
+Transformation according to the standard protocol <br />
-====<p style="font-size:15px; background-color:#66bbff;"><b>qPCR of virus stock: P816/ TKGMk-Standard; R/C: P468 from 5.10. (VLP harvested by Adrian)</b></p>====
+====<p style="font-size:15px; background-color:#66bbff;">RNA harvesting</p>====
-<b>Investigator: Achim</b><br>
-<html>
+'''Investigator: Kira <br />
+After transfection, the cells were incubated for 48 hours. Today, the cells will be harvested and RNA extracted, in order to perform RT-PCR and an additional PCR for evaluation of promoter activity.<br />
-<head>
+The transfected cells were trypsinised and centrifuged for 2 min. The supernatant was discarded and pellet washed 2x with PBS. RNeasy Kit [Qiagen] was used for RNA extraction according to the manufacturer protocol. <br />
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+c(CMV)= 335,69 ng/ul<br />
+c(P40)= 857,92 ng/ul<br />
+c(AAV_RC)= 760,21 ng/ul<br />
-<body lang=EN-US>
+===<p style="font-size:17px; background-color:#00dd77;">152. labday 17.10.2010</p>===
+====<p style="font-size:15px; background-color:#66bbff;"><b>Test digestion of pSB1C3_lITR_CMV_ß-globin_CFP</b></p>====
+<b>Investigator: Anna</b><br>
-<div class=WordSection1>
+Vector name:<br />
+pSB1C3_lITR_CMV_betaglobin_CFP_cl1 (P880): c = 452,67 ng/µl<br />
+pSB1C3_lITR_CMV_betaglobin_CFP_cl2 (P881): c = 288,88 ng/µl<br />
+pSB1C3_lITR_CMV_betaglobin_CFP_cl3 (P882): c = 288,36 ng/µl<br />
-<p class=MsoNormal><u>Protocol for quantitative real-time PCR of virus
+<b>Test Digestion:</b>
-particles</u></p>
+<br />
+{| border="1"
+| <b>components</b>   || align="right" |<b>volume P880 - P882  /µl</b> || align="right" |<b>volume P434  /µl </b>
+|-
+| DNA  ||  align="right" |2 ||  align="right" |2
+|-
+| BSA (10x) ||  align="right" |1 ||  align="right" |1
+|-
+| Buffer 4 (10x)||  align="right" |1 ||  align="right" |1
+|-
+|Enzyme NgoMIV||  align="right" |0,3||  align="right" |0,3
+|-
+|Enzyme AgeI||  align="right" |0,3||  align="right" |0,3
+|-
+|H2O||  align="right" |5,4||  align="right" |5,4
+|-
+|'''Total volume'''||  align="right" | 10||  align="right" | 10
+|}
-<p class=MsoNormal>Date: 12.10.</p>
+<br />
+<b>Gel:</b><br />
+,5 g Agarose, 50 ml TAE (1%), 3 µl GELRED , at 115 Volt<br />
-<p class=MsoNormal>Achim</p>
-<p class=MsoNormal>Virus Stock: A.F.; P816; TKGMK-Standard; R/C: P468; 5.10;
+<br/>
-consists of supernatant from pelleted cell fragments.</p>
+[[Image:Freiburg10_Test digestion of pSB1C3_lITR_CMV_ß-globin_CFP.jpg|thumb|center|350px|]]<br/>
-<p class=MsoNormal>Following the Protocol used by (Rohr et al., 2002).</p>
+====<p style="font-size:15px; background-color:#66bbff;"><b>Cloning of hGH_rITR into pSB1C3_lITR_CMV_betaglobin_CFP</b></p>====
+<b>Investigator: Stefan</b><br>
-<p class=MsoNormal>I digested 5 µl virus dilution with 7,5 µl DNAse I (7,5 units)
+<p style="font-size:13px; color:red;">Cloning of our last GOI!</p><br />
-and 25 µl 50 mM MgCl2 (end concentration 25 mM) in a final volume of 50 µl at
-°C for 30 min. </p>
-<p class=MsoListParagraph style='text-indent:-18.0pt'><span style='font-family:
+Vector name:<br />
-Wingdings'>è<span style='font:7.0pt "Times New Roman"'> </span></span>DNAse
+pSB1C3_lITR_CMV_betaglobin_CFP cl 1-3 (P880-P882)<br />
-should be heat inactivated at 65°C for 10 min. I forgot that step. The enzyme
-should however be inactivated in the initial denaturation step.</p>
-<p class=MsoNormal>I prepared the following PCR reactions:</p>
+Insert name:<br />
+pSB1C3_hGH_rITR (P728)<br />
-<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0
+<b>Digestion:</b><br /><br />
- style='border-collapse:collapse;border:none'>
- <tr>
-  <td width=71 valign=top style='width:53.3pt;border:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>Sample</p>
-  </td>
-  <td width=51 valign=top style='width:37.9pt;border:solid windowtext 1.0pt;
-  border-left:none;padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=51 valign=top style='width:37.9pt;border:solid windowtext 1.0pt;
-  border-left:none;padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>2</p>
-  </td>
-  <td width=51 valign=top style='width:37.9pt;border:solid windowtext 1.0pt;
-  border-left:none;padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>3</p>
-  </td>
-  <td width=51 valign=top style='width:37.9pt;border:solid windowtext 1.0pt;
-  border-left:none;padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>4</p>
-  </td>
-  <td width=50 valign=top style='width:37.85pt;border:solid windowtext 1.0pt;
-  border-left:none;padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>5</p>
-  </td>
-  <td width=50 valign=top style='width:37.85pt;border:solid windowtext 1.0pt;
-  border-left:none;padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>6</p>
-  </td>
-  <td width=51 valign=top style='width:38.3pt;border:solid windowtext 1.0pt;
-  border-left:none;padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>7</p>
-  </td>
-  <td width=51 valign=top style='width:38.25pt;border:solid windowtext 1.0pt;
-  border-left:none;padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>8</p>
-  </td>
-  <td width=63 valign=top style='width:47.25pt;border:solid windowtext 1.0pt;
-  border-left:none;padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>&nbsp;</p>
-  </td>
-  <td width=51 valign=top style='width:38.35pt;border:solid windowtext 1.0pt;
-  border-left:none;padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>9</p>
-  </td>
-  <td width=51 valign=top style='width:38.35pt;border:solid windowtext 1.0pt;
-  border-left:none;padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>10</p>
-  </td>
- </tr>
- <tr>
-  <td width=71 valign=top style='width:53.3pt;border:solid windowtext 1.0pt;
-  border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>&nbsp;</p>
-  </td>
-  <td width=51 valign=top style='width:37.9pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1.1</p>
-  </td>
-  <td width=51 valign=top style='width:37.9pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1.2</p>
-  </td>
-  <td width=51 valign=top style='width:37.9pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>2.1</p>
-  </td>
-  <td width=51 valign=top style='width:37.9pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>2.2</p>
-  </td>
-  <td width=50 valign=top style='width:37.85pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>S.1</p>
-  </td>
-  <td width=50 valign=top style='width:37.85pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>S.2</p>
-  </td>
-  <td width=51 valign=top style='width:38.3pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>NP1</p>
-  </td>
-  <td width=51 valign=top style='width:38.25pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>NP2</p>
-  </td>
-  <td width=63 valign=top style='width:47.25pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>MM for samples 1 -8</p>
-  </td>
-  <td width=51 valign=top style='width:38.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>NH1</p>
-  </td>
-  <td width=51 valign=top style='width:38.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>NH2</p>
-  </td>
- </tr>
- <tr>
-  <td width=71 valign=top style='width:53.3pt;border:solid windowtext 1.0pt;
-  border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>PCRmix</p>
-  </td>
-  <td width=51 valign=top style='width:37.9pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>12.5</p>
-  </td>
-  <td width=51 valign=top style='width:37.9pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>12.5</p>
-  </td>
-  <td width=51 valign=top style='width:37.9pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>12.5</p>
-  </td>
-  <td width=51 valign=top style='width:37.9pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>12.5</p>
-  </td>
-  <td width=50 valign=top style='width:37.85pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>12.5</p>
-  </td>
-  <td width=50 valign=top style='width:37.85pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>12.5</p>
-  </td>
-  <td width=51 valign=top style='width:38.3pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>12.5</p>
-  </td>
-  <td width=51 valign=top style='width:38.25pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>12.5</p>
-  </td>
-  <td width=63 valign=top style='width:47.25pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>112.5</p>
-  </td>
-  <td width=51 valign=top style='width:38.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>12.5</p>
-  </td>
-  <td width=51 valign=top style='width:38.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>12.5</p>
-  </td>
- </tr>
- <tr>
-  <td width=71 valign=top style='width:53.3pt;border:solid windowtext 1.0pt;
-  border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>Primer for</p>
-  </td>
-  <td width=51 valign=top style='width:37.9pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=51 valign=top style='width:37.9pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=51 valign=top style='width:37.9pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=51 valign=top style='width:37.9pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=50 valign=top style='width:37.85pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=50 valign=top style='width:37.85pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=51 valign=top style='width:38.3pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=51 valign=top style='width:38.25pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=63 valign=top style='width:47.25pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>9</p>
-  </td>
-  <td width=51 valign=top style='width:38.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>-</p>
-  </td>
-  <td width=51 valign=top style='width:38.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>-</p>
-  </td>
- </tr>
- <tr>
-  <td width=71 valign=top style='width:53.3pt;border:solid windowtext 1.0pt;
-  border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>Primer rev</p>
-  </td>
-  <td width=51 valign=top style='width:37.9pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=51 valign=top style='width:37.9pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=51 valign=top style='width:37.9pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=51 valign=top style='width:37.9pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=50 valign=top style='width:37.85pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=50 valign=top style='width:37.85pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=51 valign=top style='width:38.3pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=51 valign=top style='width:38.25pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=63 valign=top style='width:47.25pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>9</p>
-  </td>
-  <td width=51 valign=top style='width:38.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>-</p>
-  </td>
-  <td width=51 valign=top style='width:38.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>-</p>
-  </td>
- </tr>
- <tr>
-  <td width=71 valign=top style='width:53.3pt;border:solid windowtext 1.0pt;
-  border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>Template </p>
-  </td>
-  <td width=51 valign=top style='width:37.9pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>2</p>
-  </td>
-  <td width=51 valign=top style='width:37.9pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>2</p>
-  </td>
-  <td width=51 valign=top style='width:37.9pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>2</p>
-  </td>
-  <td width=51 valign=top style='width:37.9pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>2</p>
-  </td>
-  <td width=50 valign=top style='width:37.85pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>5</p>
-  </td>
-  <td width=50 valign=top style='width:37.85pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>5</p>
-  </td>
-  <td width=51 valign=top style='width:38.3pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>-</p>
-  </td>
-  <td width=51 valign=top style='width:38.25pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>-</p>
-  </td>
-  <td width=63 valign=top style='width:47.25pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>-</p>
-  </td>
-  <td width=51 valign=top style='width:38.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>-</p>
-  </td>
-  <td width=51 valign=top style='width:38.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>-</p>
-  </td>
- </tr>
- <tr>
-  <td width=71 valign=top style='width:53.3pt;border:solid windowtext 1.0pt;
-  border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>H2O</p>
-  </td>
-  <td width=51 valign=top style='width:37.9pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>8.5</p>
-  </td>
-  <td width=51 valign=top style='width:37.9pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>8.5</p>
-  </td>
-  <td width=51 valign=top style='width:37.9pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>8.5</p>
-  </td>
-  <td width=51 valign=top style='width:37.9pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>8.5</p>
-  </td>
-  <td width=50 valign=top style='width:37.85pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>5.5</p>
-  </td>
-  <td width=50 valign=top style='width:37.85pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>5.5</p>
-  </td>
-  <td width=51 valign=top style='width:38.3pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>10.5</p>
-  </td>
-  <td width=51 valign=top style='width:38.25pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>10.5</p>
-  </td>
-  <td width=63 valign=top style='width:47.25pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>-</p>
-  </td>
-  <td width=51 valign=top style='width:38.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>12.5</p>
-  </td>
-  <td width=51 valign=top style='width:38.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>12.5</p>
-  </td>
- </tr>
- <tr>
-  <td width=71 valign=top style='width:53.3pt;border:solid windowtext 1.0pt;
-  border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>Total</p>
-  </td>
-  <td width=51 valign=top style='width:37.9pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>25</p>
-  </td>
-  <td width=51 valign=top style='width:37.9pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>25</p>
-  </td>
-  <td width=51 valign=top style='width:37.9pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>25</p>
-  </td>
-  <td width=51 valign=top style='width:37.9pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>25</p>
-  </td>
-  <td width=50 valign=top style='width:37.85pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>25</p>
-  </td>
-  <td width=50 valign=top style='width:37.85pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>25</p>
-  </td>
-  <td width=51 valign=top style='width:38.3pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>25</p>
-  </td>
-  <td width=51 valign=top style='width:38.25pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>25</p>
-  </td>
-  <td width=63 valign=top style='width:47.25pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>&nbsp;</p>
-  </td>
-  <td width=51 valign=top style='width:38.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>25</p>
-  </td>
-  <td width=51 valign=top style='width:38.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>25</p>
-  </td>
- </tr>
-</table>
-<p class=MsoNormal>&nbsp;</p>
+<br />
+{| border="1"
+| <b>components</b>   || align="right" |<b>volume P880 - P882  /µl</b> || align="right" |<b>volume P728  /µl </b>
+|-
+| DNA  ||  align="right" |3 ||  align="right" |8
+|-
+| BSA (10x) ||  align="right" |2 ||  align="right" |2
+|-
+| Buffer 4 (10x)||  align="right" |2 ||  align="right" |2
+|-
+|Enzyme PstI||  align="right" |1||  align="right" |1
+|-
+|Enzyme XbaI||  align="right" |-||  align="right" |1
+|-
+|Enzyme SpeI||  align="right" |1||  align="right" |-
+|-
+|H2O||  align="right" |11||  align="right" |4
+|-
+|'''Total volume (e.g. 15,20,25,30 µl)'''||  align="right" | 20||  align="right" | 20
+|}
-<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0
+<br />
- style='border-collapse:collapse;border:none'>
+<b>Gel:</b><br />
- <tr>
+,5 g Agarose, 50 ml TAE (1%), 3 µl GELRED , at 115 Volt<br />
-  <td width=214 valign=top style='width:160.35pt;border:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>Step</p>
-  </td>
-  <td width=214 valign=top style='width:160.35pt;border:solid windowtext 1.0pt;
-  border-left:none;padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>Time</p>
-  </td>
-  <td width=214 valign=top style='width:160.4pt;border:solid windowtext 1.0pt;
-  border-left:none;padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>Temp</p>
-  </td>
- </tr>
- <tr>
-  <td width=214 valign=top style='width:160.35pt;border:solid windowtext 1.0pt;
-  border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>Initial denaturation</p>
-  </td>
-  <td width=214 valign=top style='width:160.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>7’</p>
-  </td>
-  <td width=214 valign=top style='width:160.4pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>95°C</p>
-  </td>
- </tr>
- <tr>
-  <td width=214 valign=top style='width:160.35pt;border:solid windowtext 1.0pt;
-  border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>&nbsp;</p>
-  </td>
-  <td width=214 valign=top style='width:160.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>&nbsp;</p>
-  </td>
-  <td width=214 valign=top style='width:160.4pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>&nbsp;</p>
-  </td>
- </tr>
- <tr>
-  <td width=214 valign=top style='width:160.35pt;border:solid windowtext 1.0pt;
-  border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>Denaturation</p>
-  </td>
-  <td width=214 valign=top style='width:160.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>10’’</p>
-  </td>
-  <td width=214 valign=top style='width:160.4pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>95°C</p>
-  </td>
- </tr>
- <tr>
-  <td width=214 valign=top style='width:160.35pt;border:solid windowtext 1.0pt;
-  border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>Annealing/Extension</p>
-  </td>
-  <td width=214 valign=top style='width:160.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>30’’</p>
-  </td>
-  <td width=214 valign=top style='width:160.4pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>60°C</p>
-  </td>
- </tr>
- <tr>
-  <td width=214 valign=top style='width:160.35pt;border:solid windowtext 1.0pt;
-  border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>Number of Cycles</p>
-  </td>
-  <td width=214 valign=top style='width:160.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>45</p>
-  </td>
-  <td width=214 valign=top style='width:160.4pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>&nbsp;</p>
-  </td>
- </tr>
-</table>
-<p class=MsoNormal>&nbsp;</p>
+<br/>
+[[Image:Freiburg10 171010.jpg|550px|thumb|center]]<br/>
-<p class=MsoNormal><u>Results:</u></p>
+<p style="font-size:13px; color:red;">Test digestion of all constructs looked alright, therefore, cloning was continued using P881 only.</p><br />
-</html>
+<br/>
-[[Image:Freiburg10 2010-10-12 qPCR Auswertung.PNG|800px|thumb|none]]
+<b>Gel extraction</b>: <br>
-<html>
+Was performed according to protocol.
-<p class=MsoListParagraphCxSpFirst style='text-indent:-18.0pt'><span
+<br>
-style='font-family:Wingdings'>è<span style='font:7.0pt "Times New Roman"'> </span></span>Both
+<b>T4 Ligation</b>: <br>
-negative controls containing primers were contaminated with DNA.</p>
+{| border="1"
+|ligation name || align="right" |728 + 881
+|-
+|volume of vector || align="right" |2,68
+|-
+|volume of insert|| align="right" |5,32
+|-
+|T4 ligase buffer (10x)|| align="right" |1
+|-
+|T4 ligase || align="right" |1
+|-
+|}
-<p class=MsoListParagraphCxSpMiddle style='text-indent:-18.0pt'><span
+<br>
-style='font-family:Wingdings'>è<span style='font:7.0pt "Times New Roman"'> </span></span>Negative
+<b>Transformation</b>: <br>
-controls containing only H2O were negative.</p>
+Transformation was performed according to standard protocol using BL21 cells.
+<br/>
-<p class=MsoListParagraphCxSpMiddle style='text-indent:-18.0pt'><span
+====<p style="font-size:15px; background-color:#66bbff;"><b>RT-PCR</b></p>====
-style='font-family:Wingdings'>è<span style='font:7.0pt "Times New Roman"'> </span></span>Values
+<b>Investigator: Kira</b><br>
-for plasmids/reaction vary; from 1 – 100 for the same virus stock</p>
+For further experiments, RNA has to be translated into cDNA. The PCR was performed according to the manufacturer protocol.
-<p class=MsoListParagraphCxSpMiddle style='text-indent:-18.0pt'><span
+===<p style="font-size:17px; background-color:#00dd77;">153. labday 18.10.2010</p>===
-style='font-family:Wingdings;color:red'>è<span style='font:7.0pt "Times New Roman"'>
+====<p style="font-size:15px; background-color:#66bbFF;"><b>quantitative real-time PCR for detection of virus titer</b></p>====
-</span></span>Dilution of the virus solution: 1:10; 2µl PCR sample: <b><u><span
+'''Investigator: Achim <br>
-style='color:red'>3.2 * 10^5 virus particles/ml</span></u></b></p>
-<p class=MsoListParagraphCxSpLast style='text-indent:-18.0pt'><span
+*qPCR of harvested virus particles to determine the virus titers of our different constructs
-style='font-family:Wingdings'>è<span style='font:7.0pt "Times New Roman"'> </span></span>Because
-of the contaminated negative controls and the high deviation, I will repeat the
-experiment tomorrow to verify the results.</p>
-<p class=MsoNormal><span lang=DE>References:</span></p>
+*Total number of samples: 58
-<p><span lang=DE style='font-size:11.0pt;font-family:"Calibri","sans-serif"'>Rohr,
+====<p style="font-size:15px; background-color:#66bbFF;"><b>Test-digestion of pSB1C3_lITR_phTERT_betaglobin_mGMK_TK30_hGH_rITR_SDM-PstI clone 1, 2 and 3 (P877, 878 and 879) </b></p>====
-U., Wulf, M., Stahn, S., Steidl, U., Haas, R., Kronenwett, R., et al. </span><span
+'''Investigator Patrick <br>
-style='font-size:11.0pt;font-family:"Calibri","sans-serif"'>(2002). Fast and
-reliable titration of recombinant adeno-associated virus type-2 using
-quantitative real-time PCR. <i>Journal of virological methods</i>, <i>106</i>(1),
--8. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/12367732.</span></p>
-<p class=MsoNormal>&nbsp;</p>
+Check the plasmid for leftITR:
+,5 µl EcoRI, 1 µl BstEII, 7 µl DNA, 2 µl Buffer 4, 2 µl BSA, 7,5 µl H2O, 45 minutes 37°C, 45 minutes 60°C
+<br>
+<br>
+Check the plasmid for hGH_rITR and ... :
+,5 µl AgeI, 0,5 µl PstI, 3 µl DNA, 1 µl BSA, 1 µl Buffer 4, 4 µl H2O, 70 minutes 37°C
-</div>
+Expected results:
+* leftITR: about 190 bp
+* hGH_rITR: about 670 bp
-</body>
+<br>
+Unfortunately the digestions had to be reapeated because i didnt switch on the current so the samples and especially the 1kb GeneRuler marker diffused.
+<br>
+<br>
+The second run: see above <br>
+[[Image:Freiburg10_1019pat_test1bfertig.jpg]]
+<br>
+PstI or BstEII seems to work not properly
-</html>
+====<p style="font-size:15px; background-color:#66bbFF;"><b>MTT Assay </b></p>====
+'''Investigator Kerstin, Anissa <br>
-====<p style="font-size:15px; background-color:#66bbff;"><b>Cloning sr39 into mGMK_TK30</b></p>====
+[[Image:Freiburg10 transductionplan 18.10..jpg]]
-<b>Investigator: Bea</b><br>
-<p style="color:#66bbff;"><i>Comment</i>: We do not only want to submit the tk30, a mutant version of the thymidine kinase, but as well another mutant protein which is named sr39. SR39 seems to have a better activity than the tk30 without being fused to the guanylate kinase (mgmk), but the sr39 also works quite well as a fusion protein. We ordered one part of the sr39 and we are now subcloning it into the mgmnk_tk30 construct in order to obtain mgmk_sr39. </p>
-<b>Protocol:</b>
-<br />
-[[Image: Freiburg10_SR39_into_mGMK_tk30_12.10.PNG|thumb|center|600px]]
-<br />
-The digested fragments were loaded on a 1.2% agarose gel. The results can be seen above in the gel picture: I cut out the small visible band in the right lane, and the upper intensive band of the left lane. <br />
-<br />
-<b>Result: </b>
-<br />
-[[Image: Freiburg10_Digest_sr39_and_mGMK_tk30.png|thumb|center|400px]]
-<br />
-After gel extraction has been performed, I ligated the two fragments and transformed BL-21 cells, plated them on cm agar plates and incubated them over night at 37°C.
-====<p style="font-size:15px; background-color:#66bbff;"><b>Concentration of virus stock for Western Blot analysis</b></p>====
+[[Image:Freiburg10 transductionplan1 18.10..jpg|thumb|center|920px]]
-<b>Investigator: Hanna </b> <br/>
-<br/>
-For the first Western Blot try, we investigate whether is is enough to concentrate the virus stock (from cell supernatant) and then to simply load it onto a SDS-PAGE gel and perfom immuno-staining. <br/>
-For this purpose 8 mL virus stock (pHelper, pAAV_RC, GOI=YFP) were concentrated via amicon filtration. <br/>
-* First, amicon filter was washed with 4 mL PBS.
-* Then 4 mL virus stock was loaded onto the filter and centrifuged at 3000 rpm for 15 minutes.
-* Flow-through was discarded and additional virus stock was loaded, centrifuged at 3000 rpm for 25 minutes.
-* Flow-through was discarded, remaining solution was re-suspended and additional virus stock was added. Amicon filter was centrifuged at 3000 rpm for 50 minutes.
-* Flow-through was discarded. Residual protein solution: 500 µL. Filter was washed with 3 mL PBS, centrifuging 35 minutes two times.
-* 2 x 40 µL protein solution (16x) was taken and mixed with Laemmli buffer and stored over night @ 4°C.
-<br/>
-<b>To do:</b> SDS-PAGE and Western Blot tomorrow. <br/>
-Perfomance of another try by harvesting viruses of the cell pellet with RIPA buffer & freeze/ thaw. <br/>
-Western Blot of unmodified, and modified capsids (N-terminal fusion and VP1 insertion with CFP ref. mVenus) --> size shift should be detectable.<br/>
-====<p style="font-size:15px; background-color:#66bbff;"><b>Transduction of HT1080 and A431</b></p>====
+[[Image:Freiburg10 transductionplan2 18.10..jpg|thumb|center|920px]]
-<b>Investigator: Kerstin </b> <br/>
+'''Results:'''
-[[Image:Freiburg10 Transductionplan 11.10.2010.jpg|700px|]]<br/>
+[[Image:Freiburg10 Results of MTTAssay 18 10 10 2day 1pic.jpg|thumb|center|920px]]
-[[Image:Freiburg10 Transductionplan1 11.10.2010.jpg|700px|]]<br/>
-[[Image:Freiburg10 Transductionplan2 11.10.2010.jpg|700px|]]<br/>
+[[Image:Freiburg10 Results of MTTAssay 18 10 10 2day 3pic.jpg|thumb|center|920px]]
-[[Image:Freiburg10 Transductionplan3 11.10.2010.jpg|700px|]]<br/>
-[[Image:Freiburg10 Transductionplan4 11.10.2010.jpg|700px|]]<br/>
+[[Image:Freiburg10 Results of MTTAssay 18 10 10 2day 2pic.jpg|thumb|center|920px]]
 ====<p style="font-size:15px; background-color:#66bbFF;"><b>Mini-Prep and test digestion of several constructs</b></p>====
-<b>Investigator: Stefan</b><br />
+<b>Investigator: Jessica</b><br />
 Glycerol stocks were prepared:<br>
 <ul>
-<li>B651 = pSB1C3_P40_VP123</li>
+<li>B702 = pSB1C3_lITR_pTERT_ßglobin_CD_hGH_rITR clone 1</li>
-<li>B652 = pSB1C3_lITR_phTERT_ß-globin_mGMK_TK30_PstI_SDM_hgH_rITR</li>
+<li>B703 = pSB1C3_lITR_pTERT_ßglobin_CD_hGH_rITR clone 2</li>
-<li>B653 = pSB1C3_lITR_phTERT_ß-globin_mGMK_TK30_PstI_SDM_hgH_rITR</li>
+<li>B704 = pSB1C3_001_VP3 clone 1</li>
-<li>B654 = pSB1C3_lITR_CMV_ß-globin_mGMK_TK30_hgH_rITR</li>
+<li>B705 = pSB1C3_001_VP3 clone 2</li>
-<li>B655 = pSB1C3_lITR_CMV_ß-globin_mGMK_TK30_hgH_rITR</li>
+<li>B706 = pSB1C3_001_VP2 clone 1</li>
-<li>B656 = pSB1C3_CMV_VP123_453_Z34C_HSPG-KO</li>
+<li>B707 = pSB1C3_001_VP2 clone 2</li>
-<li>B657 = pSB1C3_CMV_VP123_453_Z34C_HSPG-KO</li>
+<li>B708 = pSB1C3_001_Rep78 clone 1</li>
-<li>B658 = pSB1C3_CMV_VP123_453_RGD_HSPG-KO</li>
+<li>B709 = pSB1C3_001_Rep78 clone 2</li>
-<li>B659 = pSB1C3_CMV_VP123_453_RGD_HSPG-KO</li>
+<li>B710 = pSB1C3_001_Rep52 clone 1</li>
-<li>B660 = pSB1C3_lITR_phTERT_ß-globin_CFP_hgH-rITR</li>
+<li>B711 = pSB1C3_001_Rep52 clone 2</li>
-<li>B661 = pSB1C3_lITR_phTERT_ß-globin_CFP_hgH-rITR</li>
+<li>B712 = pSB1C3_lITR_CMV_ßglobin_CD_hGH_rITR clone 1</li>
+<li>B713 = pSB1C3_lITR_CMV_ßglobin_CD_hGH_rITR clone 2</li>
+<li>B714 = pSB1C3_001_VP1 clone 1</li>
+<li>B715 = pSB1C3_001_VP1 clone 2</li>
 </ul><br>
@@ Line 1,169: / Line 546: @@
 <ul>
-<li>P821 = pSB1C3_P40_VP123 c = 327,4 ng/µl</li>
+<li>P886 = pSB1C3_lITR_pTERT_ßglobin_CD_hGH_rITR clone 1 c= 232,2ng/µl</li>
-<li>P822 = pSB1C3_lITR_phTERT_ß-globin_mGMK_TK30_pSTI_SDM_hgH_rITR c = 342,9 ng/µl</li>
+<li>P887 = pSB1C3_lITR_pTERT_ßglobin_CD_hGH_rITR clone 2 c= 186,2ng/µl</li>
-<li>P823 = pSB1C3_lITR_phTERT_ß-globin_mGMK_TK30_pSTI_SDM_hgH_rITR c = 324,0 ng/µl</li>
+<li>P888 = pSB1C3_001_VP3 clone 1 c= 300,8ng/µl</li>
-<li>P824 = pSB1C3_lITR_CMV_ß-globin_mGMK_TK30_hgH_rITR c = 81,6 ng/µl</li>
+<li>P889 = pSB1C3_001_VP3 clone 2 c= 284,4ng/µl</li>
-<li>P825 = pSB1C3_lITR_CMV_ß-globin_mGMK_TK30_hgH_rITR c = 148,4 ng/µl</li>
+<li>P890 = pSB1C3_001_VP2 clone 1 c= 298,3ng/µl</li>
-<li>P826 = pSB1C3_CMV_VP123_453_Z34C_HSPG-KO c = 105,8 ng/µl</li>
+<li>P891 = pSB1C3_001_VP2 clone 2 c= 299,9ng/µl</li>
-<li>P827 = pSB1C3_CMV_VP123_453_Z34C_HSPG-KO c = 121,1 ng/µl</li>
+<li>P892 = pSB1C3_001_Rep78 clone 1 c= 143,9ng/µl</li>
-<li>P828 = pSB1C3_CMV_VP123_453_RGD_HSPG-KO c = 188,4 ng/µl</li>
+<li>P893 = pSB1C3_001_Rep78 clone 2 c= 163,4ng/µl</li>
-<li>P829 = pSB1C3_CMV_VP123_453_RGD_HSPG-KO c = 140,4 ng/µl</li>
+<li>P894 = pSB1C3_001_Rep52 clone 1 c= 166,6ng/µl</li>
-<li>P830 = pSB1C3_lITR_phTERT_ß-globin_CFP_hgH-rITR c = 197,2 ng/µl</li>
+<li>P895 = pSB1C3_001_Rep52 clone 2 c= 181,6ng/µl</li>
-<li>P831 = pSB1C3_lITR_phTERT_ß-globin_CFP_hgH-rITR c = 207,3 ng/µl</li>
+<li>P896 = pSB1C3_lITR_CMV_ßglobin_CD_hGH_rITR clone 1 c= 250,5ng/µl</li>
+<li>P897 = pSB1C3_lITR_CMV_ßglobin_CD_hGH_rITR clone 2 c= 173,4ng/µl</li>
+<li>P898 = pSB1C3_001_VP1 clone 1 c= 272,7ng/µl</li>
+<li>P899 = pSB1C3_001_VP1 clone 2 c= 294,8ng/µl</li>
+<li>P900 = pSB1C3_hGH_rITR (from B160) c= 136,7ng/µl</li>
 </ul>
 Test digestion:<br>
 {| border="1"
-| align="left" | '''Components''' ||align="left"| <b>P822 + P823 / µl</b>||align="left"| <b>P824 + P825 / µl</b>||align="left"| <b>P830 + P831 / µl</b>
+| align="left" | '''Components''' ||align="left"| <b>P886,887,892,893,894,895,896,897 / µl</b>||align="left"| <b>P888,889,890,891898,899 / µl</b>
 |-
-| align="left" | DNA   ||align="left"| 1,5||align="left"| 1,5||align="left"| 2
+| align="left" | DNA   ||align="left"| 1,5||align="left"| 1,5
 |-
-| align="left" | Buffer 4 ||align="left"| 1||align="left"| 1||align="left"| 1
+| align="left" | Buffer  ||align="left"| (4) 1||align="left"| (2) 1
 |-
-| align="left" | BSA (10x)  ||align="left"| 1||align="left"| 1||align="left"| 1
+| align="left" | BSA (10x)  ||align="left"| 1||align="left"| 1
 |-
-| align="left" | PstI ||align="left"| 0,3 ||align="left"| - ||align="left"| -
+| align="left" | NgoMIV ||align="left"| 0,4 ||align="left"| -
 |-
-| align="left" | XbaI ||align="left"| 0,3 ||align="left"| 0,3 ||align="left"| 0,3
+| align="left" | XbaI ||align="left"| 0,4 ||align="left"| -
 |-
-| align="left" | SpeI ||align="left"| - ||align="left"| 0,3 ||align="left"| 0,3
+| align="left" | PstI ||align="left"| - ||align="left"| 0,6
 |-
-| align="left" | H<sub>2</sub>O ||align="left"| 5,9||align="left"| 5,9||align="left"| 5,4
+| align="left" | XcmI ||align="left"| - ||align="left"| 0,4
 |-
-| align="left" | '''Total volume''' ||align="left"| <b>10</b>  ||align="left"| <b>10</b> ||align="left"| <b>10</b>
+| align="left" | H<sub>2</sub>O ||align="left"| 4,5||align="left"| 4,5
+|-
+| align="left" | '''Total volume''' ||align="left"| <b>10</b>  ||align="left"| <b>10</b>
 |}
 Gel:<br>
-,5g agarose, 50 ml TAE (1%), 3 µl GELRED, 115 Volt, running time ~50 minutes<br>
+,0g agarose, 100 ml TAE (1%), 6 µl GELRED,  Volt, running time  minutes<br>
-[[Image:Freiburg10 test digestion 121010 2.jpg|550px]]
+[[Image:Freiburg10 test digestion 886-899.jpg|thumb|center|800px]]
-<p style="color:#66bbff;"><i>Comment</i>: P822 and P823 smear a lot, therefore evaluation is difficult. Another test digestion would be reasonable.<br> P824 contains hgH_rITR, therefore it will be discarded. P825 looks well and was sent for sequencing.<br>
+<p style="color:#66bbff;"><i>Comment</i>: Rep 52/78 will be checked,pSB1C3_lITR_CMV_ßglobin_CD_hGH_rITR and pSB1C3_lITR_pTERT_ßglobin_CD_hGH_rITR will be sequenced</p>
-P830 and P831 look well and will be sent for sequencing tomorrow.</p>
-====<p style="font-size:15px; background-color:#66bbFF;"><b>Repetiton: Site-directed mutagenesis in pSB1C3_CD construct</b></p>====
+====<p style="font-size:15px; background-color:#66bbff;"><b>PCR of mGMK and SR39</b></p>====
-<b>Investigator: Stefan</b>
+<b>Investigator: Anna</b><br>
-<br/>
-<p style="color:#66bbff;"><i>Comment</i>: Sequencing revealed that there is no CD in our pSB1C3_CD but rather CFP. So, anonther approach of the SDM needs to be performed.</p>
-<b>Protocol using the QuickChange Lightning Kit from Stratagene: </b>
+Plasmids:<br />
+pSB1C3_mGMK_TK30_SDM-PstI clone 2(P804)<br />
+pSB1C3_mGMK_sr39 clone 1(P860)<br />
+Oligos:<br />
+O193: pTK30_for<br />
+O81: pmgmk_tk30_suffix_RFC25_rev
 <br />
+<b>PCR Mix:</b>
+{| border="1"
+| <b>Components</b>   || align="right" |<b>Volume  /µl</b>
+|-
+| Phusion Buffer  ||  align="right" |10
+|-
+| dNTP ||  align="right" |1
+|-
+|Primer_for||  align="right" |2,5
+|-
+|Primer_rev||  align="right" |2,5
+|-
+|DNA template||  align="right" |1
+|-
+|H2O||  align="right" |32,5
+|-
+|'''Total volume'''||  align="right" | 50
+|}
 <br />
-<b>Ingredients: </b>
+<b>PCR Program:</b>
-<br>
 {| border="1"
-| '''Ingredients''' || align="right" |Volume / µl
+|Cycles||Temperature||Time
 |-
-| 10x reaction buffer ||  align="right" |2,5
+|||98°C||60 sec
 |-
-| dNTP||  align="right" |0,5
+|||98°C||15 sec
 |-
-| forward primer: O191 ||  align="right" |0,56
+|8x||52°C||25 sec
 |-
-| reverse primer: O192 ||  align="right" |0,57
+|||72°C||25 sec
 |-
-| DNA Template||  align="right" |0,5 (1:10 dilution)
+|||98°C||15 sec
 |-
-| QuikSolution Reagent||  align="right" |0,75
+|17x||67°C||25 sec
 |-
-|QuikChangeLightning Enzyme||  align="right" |0,5
+|||72°C||25 sec
 |-
-|H<sub>2</sub>O||  align="right" |14,35
+|1x||72°C||5 min
 |-
-|Total volume||  align="right" |25
+|Hold 4°C
 |}
 <br />
-<br />
+<b>Gel:</b><br />
-The following PCR program was used:
+,5 g Agarose, 50 ml TAE (1%), 3 µl GELRED , at 115 Volt<br />
-<ul>
-<li>95 °C 120 s</li>
-<li>95 °C 20 s</li>
-<li>60 °C 60 s</li>
-<li>68 °C 105 s</li>
-<li>Goto step 2 repeat 17 times</li>
-<li>68 °C 300 s</li>
-</ul>
-<b>Digestion using DpnI:</b><br/>
-To digest methylated and hemi-methylated DNA, 1 µl of DpnI was added and the mixture was incubated for 10 minutes at 37 °C. <br/>
-<b>Transformation:</b><br/>
-Trafo was performed according to protocol, but using XL1b cells instead of XL-10 Gold cells.
-===<p style="font-size:17px; background-color:#00dd77;">148. labday 13.10.2010</p>===
-====<p style="font-size:15px; background-color:#66bbFF;"><b>qPCRs of virus stocks</b></p>====
-<b>Investigator: Achim</b>
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-	font-family:"Calibri","sans-serif";}
-p.MsoAcetate, li.MsoAcetate, div.MsoAcetate
-	{mso-style-link:"Sprechblasentext Zchn";
-	margin:0cm;
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-	{mso-style-name:"Kopfzeile Zchn";
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-	{mso-style-name:"Sprechblasentext Zchn";
-	mso-style-link:Sprechblasentext;
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-.MsoChpDefault
-	{font-family:"Calibri","sans-serif";}
-.MsoPapDefault
-	{margin-bottom:10.0pt;
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- /* Page Definitions */
- @page WordSection1
-	{size:792.0pt 612.0pt;
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--->
-</style>
-</head>
-<body lang=EN-US>
-<div class=WordSection1>
-<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0
- style='border-collapse:collapse;border:none'>
- <tr>
-  <td width=450 valign=top style='width:337.6pt;border:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>Sample:</p>
-  </td>
-  <td width=181 valign=top style='width:135.6pt;border:solid windowtext 1.0pt;
-  border-left:none;padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>Plasmid, Glycerol stock:</p>
-  </td>
-  <td width=181 valign=top style='width:135.6pt;border:solid windowtext 1.0pt;
-  border-left:none;padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>Sample No.</p>
-  </td>
- </tr>
- <tr>
-  <td width=450 valign=top style='width:337.6pt;border:solid windowtext 1.0pt;
-  border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'><span style='color:black'>pSB1C3_001_RC_IRCK_HSPG-ko_P5tataless_RFC10
-  clone 1</span></p>
-  </td>
-  <td width=181 valign=top style='width:135.6pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>B561</p>
-  </td>
-  <td width=181 valign=top style='width:135.6pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
- </tr>
- <tr>
-  <td width=450 valign=top style='width:337.6pt;border:solid windowtext 1.0pt;
-  border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'><span style='font-size:10.0pt;color:black'>pSB1C3_001_RC_IRCK_P5tataless
-  clone1</span></p>
-  </td>
-  <td width=181 valign=top style='width:135.6pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>P668</p>
-  </td>
-  <td width=181 valign=top style='width:135.6pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>2</p>
-  </td>
- </tr>
- <tr>
-  <td width=450 valign=top style='width:337.6pt;border:solid windowtext 1.0pt;
-  border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'><span style='font-size:10.0pt;color:black'>pSB1C3_lITR_CMV_beta-globin_mVenus_rITR
-  clone1</span></p>
-  </td>
-  <td width=181 valign=top style='width:135.6pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>P378</p>
-  </td>
-  <td width=181 valign=top style='width:135.6pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>3</p>
-  </td>
- </tr>
- <tr>
-  <td width=450 valign=top style='width:337.6pt;border:solid windowtext 1.0pt;
-  border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'><span style='font-size:10.0pt;color:black'>pSB1C3_lITR_CMV_mVenus_hGH_rITR
-  clone1</span></p>
-  </td>
-  <td width=181 valign=top style='width:135.6pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>P377</p>
-  </td>
-  <td width=181 valign=top style='width:135.6pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>4</p>
-  </td>
- </tr>
- <tr>
-  <td width=450 valign=top style='width:337.6pt;border:solid windowtext 1.0pt;
-  border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'><b><span style='font-size:10.0pt;color:black'>pAAV_RC_RepCapIns_SDMKpnI</span></b></p>
-  </td>
-  <td width=181 valign=top style='width:135.6pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>P468</p>
-  </td>
-  <td width=181 valign=top style='width:135.6pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>5</p>
-  </td>
- </tr>
-</table>
-<p class=MsoNormal>                                                                                                </p>
-<p class=MsoNormal>Digestion: 37°C, 30 min</p>
-<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0
- style='border-collapse:collapse;border:none'>
- <tr>
-  <td width=134 valign=top style='width:100.75pt;border:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>&nbsp;</p>
-  </td>
-  <td width=128 valign=top style='width:96.2pt;border:solid windowtext 1.0pt;
-  border-left:none;padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=128 valign=top style='width:96.15pt;border:solid windowtext 1.0pt;
-  border-left:none;padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>2</p>
-  </td>
-  <td width=128 valign=top style='width:96.15pt;border:solid windowtext 1.0pt;
-  border-left:none;padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>3</p>
-  </td>
-  <td width=128 valign=top style='width:96.15pt;border:solid windowtext 1.0pt;
-  border-left:none;padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>4</p>
-  </td>
-  <td width=127 valign=top style='width:95.4pt;border:solid windowtext 1.0pt;
-  border-left:none;padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>5</p>
-  </td>
-  <td width=126 valign=top style='width:94.45pt;border:solid windowtext 1.0pt;
-  border-left:none;padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>MM x6</p>
-  </td>
- </tr>
- <tr>
-  <td width=134 valign=top style='width:100.75pt;border:solid windowtext 1.0pt;
-  border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>Virus solution</p>
-  </td>
-  <td width=128 valign=top style='width:96.2pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>5</p>
-  </td>
-  <td width=128 valign=top style='width:96.15pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>5</p>
-  </td>
-  <td width=128 valign=top style='width:96.15pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>5</p>
-  </td>
-  <td width=128 valign=top style='width:96.15pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>5</p>
-  </td>
-  <td width=127 valign=top style='width:95.4pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>5</p>
-  </td>
-  <td width=126 valign=top style='width:94.45pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>&nbsp;</p>
-  </td>
- </tr>
- <tr>
-  <td width=134 valign=top style='width:100.75pt;border:solid windowtext 1.0pt;
-  border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>DNAseI (7,5 U)</p>
-  </td>
-  <td width=128 valign=top style='width:96.2pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>7,5</p>
-  </td>
-  <td width=128 valign=top style='width:96.15pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>7,5</p>
-  </td>
-  <td width=128 valign=top style='width:96.15pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>7,5</p>
-  </td>
-  <td width=128 valign=top style='width:96.15pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>7,5</p>
-  </td>
-  <td width=127 valign=top style='width:95.4pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>7,5</p>
-  </td>
-  <td width=126 valign=top style='width:94.45pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>45</p>
-  </td>
- </tr>
- <tr>
-  <td width=134 valign=top style='width:100.75pt;border:solid windowtext 1.0pt;
-  border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>MgCl2 50 mM</p>
-  </td>
-  <td width=128 valign=top style='width:96.2pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>5</p>
-  </td>
-  <td width=128 valign=top style='width:96.15pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>5</p>
-  </td>
-  <td width=128 valign=top style='width:96.15pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>5</p>
-  </td>
-  <td width=128 valign=top style='width:96.15pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>5</p>
-  </td>
-  <td width=127 valign=top style='width:95.4pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>5</p>
-  </td>
-  <td width=126 valign=top style='width:94.45pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>30</p>
-  </td>
- </tr>
- <tr>
-  <td width=134 valign=top style='width:100.75pt;border:solid windowtext 1.0pt;
-  border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>H2O</p>
-  </td>
-  <td width=128 valign=top style='width:96.2pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>32.5</p>
-  </td>
-  <td width=128 valign=top style='width:96.15pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>32.5</p>
-  </td>
-  <td width=128 valign=top style='width:96.15pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>32.5</p>
-  </td>
-  <td width=128 valign=top style='width:96.15pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>32.5</p>
-  </td>
-  <td width=127 valign=top style='width:95.4pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>32.5</p>
-  </td>
-  <td width=126 valign=top style='width:94.45pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>195</p>
-  </td>
- </tr>
- <tr>
-  <td width=134 valign=top style='width:100.75pt;border:solid windowtext 1.0pt;
-  border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>Total</p>
-  </td>
-  <td width=128 valign=top style='width:96.2pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>50</p>
-  </td>
-  <td width=128 valign=top style='width:96.15pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>50</p>
-  </td>
-  <td width=128 valign=top style='width:96.15pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>50</p>
-  </td>
-  <td width=128 valign=top style='width:96.15pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>50</p>
-  </td>
-  <td width=127 valign=top style='width:95.4pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>50</p>
-  </td>
-  <td width=126 valign=top style='width:94.45pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>300</p>
-  </td>
- </tr>
-</table>
-<p class=MsoNormal>&nbsp;</p>
-<p class=MsoNormal>PCR reaction:</p>
-<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 width=861
- style='width:645.85pt;border-collapse:collapse;border:none'>
- <tr style='height:13.75pt'>
-  <td width=71 valign=top style='width:53.2pt;border:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>Sample</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border:solid windowtext 1.0pt;
-  border-left:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border:solid windowtext 1.0pt;
-  border-left:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>2</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border:solid windowtext 1.0pt;
-  border-left:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>3</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border:solid windowtext 1.0pt;
-  border-left:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>4</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border:solid windowtext 1.0pt;
-  border-left:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>5</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border:solid windowtext 1.0pt;
-  border-left:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>6</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border:solid windowtext 1.0pt;
-  border-left:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>7</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border:solid windowtext 1.0pt;
-  border-left:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>8</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border:solid windowtext 1.0pt;
-  border-left:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>9</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border:solid windowtext 1.0pt;
-  border-left:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>10</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border:solid windowtext 1.0pt;
-  border-left:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>11</p>
-  </td>
-  <td width=42 valign=top style='width:31.65pt;border:solid windowtext 1.0pt;
-  border-left:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>12</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border:solid windowtext 1.0pt;
-  border-left:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>13</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border:solid windowtext 1.0pt;
-  border-left:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>14</p>
-  </td>
-  <td width=115 valign=top style='width:85.95pt;border:solid windowtext 1.0pt;
-  border-left:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>&nbsp;</p>
-  </td>
-  <td width=47 valign=top style='width:35.4pt;border:solid windowtext 1.0pt;
-  border-left:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>15</p>
-  </td>
-  <td width=60 valign=top style='width:45.1pt;border:solid windowtext 1.0pt;
-  border-left:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>16</p>
-  </td>
- </tr>
- <tr style='height:41.2pt'>
-  <td width=71 valign=top style='width:53.2pt;border:solid windowtext 1.0pt;
-  border-top:none;padding:0cm 5.4pt 0cm 5.4pt;height:41.2pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>&nbsp;</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:41.2pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1.1</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:41.2pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1.2</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:41.2pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>2.1</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:41.2pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>2.2</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:41.2pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>3.1</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:41.2pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>3.2</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:41.2pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>4.1</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:41.2pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>4.2</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:41.2pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>5.1</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:41.2pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>5.2</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:41.2pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>S.1</p>
-  </td>
-  <td width=42 valign=top style='width:31.65pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:41.2pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>S.2</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:41.2pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>NP1</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:41.2pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>NP2</p>
-  </td>
-  <td width=115 valign=top style='width:85.95pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:41.2pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>MM for samples 1 -14 (x15)</p>
-  </td>
-  <td width=47 valign=top style='width:35.4pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:41.2pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>NH1</p>
-  </td>
-  <td width=60 valign=top style='width:45.1pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:41.2pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>NH2</p>
-  </td>
- </tr>
- <tr style='height:12.85pt'>
-  <td width=71 valign=top style='width:53.2pt;border:solid windowtext 1.0pt;
-  border-top:none;padding:0cm 5.4pt 0cm 5.4pt;height:12.85pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>PCRmix</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:12.85pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>12.5</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:12.85pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>12.5</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:12.85pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>12.5</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:12.85pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>12.5</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:12.85pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>12.5</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:12.85pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>12.5</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:12.85pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>12.5</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:12.85pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>12.5</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:12.85pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>12.5</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:12.85pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>12.5</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:12.85pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>12.5</p>
-  </td>
-  <td width=42 valign=top style='width:31.65pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:12.85pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>12.5</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:12.85pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>12.5</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:12.85pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>12.5</p>
-  </td>
-  <td width=115 valign=top style='width:85.95pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:12.85pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>187,5</p>
-  </td>
-  <td width=47 valign=top style='width:35.4pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:12.85pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>12.5</p>
-  </td>
-  <td width=60 valign=top style='width:45.1pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:12.85pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>12.5</p>
-  </td>
- </tr>
- <tr style='height:27.45pt'>
-  <td width=71 valign=top style='width:53.2pt;border:solid windowtext 1.0pt;
-  border-top:none;padding:0cm 5.4pt 0cm 5.4pt;height:27.45pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>Primer for 1:10</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:27.45pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:27.45pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:27.45pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:27.45pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:27.45pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:27.45pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:27.45pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:27.45pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:27.45pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:27.45pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:27.45pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=42 valign=top style='width:31.65pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:27.45pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:27.45pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:27.45pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=115 valign=top style='width:85.95pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:27.45pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>15</p>
-  </td>
-  <td width=47 valign=top style='width:35.4pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:27.45pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>-</p>
-  </td>
-  <td width=60 valign=top style='width:45.1pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:27.45pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>-</p>
-  </td>
- </tr>
- <tr style='height:27.45pt'>
-  <td width=71 valign=top style='width:53.2pt;border:solid windowtext 1.0pt;
-  border-top:none;padding:0cm 5.4pt 0cm 5.4pt;height:27.45pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>Primer rev 1:10</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:27.45pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:27.45pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:27.45pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:27.45pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:27.45pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:27.45pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:27.45pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:27.45pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:27.45pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:27.45pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:27.45pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=42 valign=top style='width:31.65pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:27.45pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:27.45pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:27.45pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>1</p>
-  </td>
-  <td width=115 valign=top style='width:85.95pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:27.45pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>15</p>
-  </td>
-  <td width=47 valign=top style='width:35.4pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:27.45pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>-</p>
-  </td>
-  <td width=60 valign=top style='width:45.1pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:27.45pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>-</p>
-  </td>
- </tr>
- <tr style='height:12.85pt'>
-  <td width=71 valign=top style='width:53.2pt;border:solid windowtext 1.0pt;
-  border-top:none;padding:0cm 5.4pt 0cm 5.4pt;height:12.85pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>Template </p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:12.85pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>2</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:12.85pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>2</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:12.85pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>2</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:12.85pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>2</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:12.85pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>2</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:12.85pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>2</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:12.85pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>2</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:12.85pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>2</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:12.85pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>2</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:12.85pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>2</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:12.85pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>5</p>
-  </td>
-  <td width=42 valign=top style='width:31.65pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:12.85pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>5</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:12.85pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>-</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:12.85pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>-</p>
-  </td>
-  <td width=115 valign=top style='width:85.95pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:12.85pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>-</p>
-  </td>
-  <td width=47 valign=top style='width:35.4pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:12.85pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>-</p>
-  </td>
-  <td width=60 valign=top style='width:45.1pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:12.85pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>-</p>
-  </td>
- </tr>
- <tr style='height:13.75pt'>
-  <td width=71 valign=top style='width:53.2pt;border:solid windowtext 1.0pt;
-  border-top:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>H2O</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>8.5</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>8.5</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>8.5</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>8.5</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>8.5</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>8.5</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>8.5</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>8.5</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>8.5</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>8.5</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>5.5</p>
-  </td>
-  <td width=42 valign=top style='width:31.65pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>5.5</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>10.5</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>10.5</p>
-  </td>
-  <td width=115 valign=top style='width:85.95pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>-</p>
-  </td>
-  <td width=47 valign=top style='width:35.4pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>12.5</p>
-  </td>
-  <td width=60 valign=top style='width:45.1pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>12.5</p>
-  </td>
- </tr>
- <tr style='height:13.75pt'>
-  <td width=71 valign=top style='width:53.2pt;border:solid windowtext 1.0pt;
-  border-top:none;padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>Total</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>25</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>25</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>25</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>25</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>25</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>25</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>25</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>25</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>25</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>25</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>25</p>
-  </td>
-  <td width=42 valign=top style='width:31.65pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>25</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>25</p>
-  </td>
-  <td width=40 valign=top style='width:30.35pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>25</p>
-  </td>
-  <td width=115 valign=top style='width:85.95pt;border-top:none;border-left:
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>&nbsp;</p>
-  </td>
-  <td width=47 valign=top style='width:35.4pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>25</p>
-  </td>
-  <td width=60 valign=top style='width:45.1pt;border-top:none;border-left:none;
-  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-  padding:0cm 5.4pt 0cm 5.4pt;height:13.75pt'>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;line-height:
-  normal'>25</p>
-  </td>
- </tr>
-</table>
-<p class=MsoNormal>&nbsp;</p>
-</div>
-</body>
-</html>
-Results:
-[[Image:Freiburg10 13 10 auswertung.PNG|thumb|none|800px]]
-====<p style="font-size:15px; background-color:#66bbFF;"><b>SDS Page of unmodified AAV2 capsids</b></p>====
-<b>Investigator: Hanna</b>
 <br/>
+[[Image:|550px|]]<br/>
-<b>Comment:</b> Yesterday a 8x concentration of AAV2 supernatant-stock was performed. 10 µL Laemmli-buffer was added to 40 µL AAV stock (8x) and stored over night at 4°C. <br/>
+===<p style="font-size:17px; background-color:#00dd77;">154. labday 19.10.2010</p>===
-Today a SDS-PAGE of these samples will be performed. <br/>
-<br/>
-Samples were incubated for 10 minutes at 95°C for denaturation. <br/>
-<br/>
-<b>Loading plan:</b>
-{| border="1"
-| align="left" | '''5 µL PageRuler Prestained Protein Ladder''' ||align="left"| <b>10 µL Sample</b>||align="left"| <b>15 µL Sample</b>||align="left"| <b>25 µL Sample</b>
-|}
-<br/>
-After running front reached the end of the gel, PAGE was stopped. <br/>
-<br/>
-<b>Western Blot:</b> <br/>
-Three layers of transfer buffer soaked whatman paper were placed onto the blotter. A polyvinylidene difluoride membrane was activated by methanol (5 seconds) and then incubated for 5 minutes in transfer buffer. Membrane was then placed onto the whatman paper, gel was carefully layed on top. Air bubbles were removed, ensuring elictricity flow. After coverage by 3 further layers of whatman, blotting was performed at 200 mA for 1 hour. <br/>
-<br/>
-<b>Blocking:</b> <br/>
-In order to minimize nonspecific antibody interactions, membrane was incubated in blocking buffer (TBS (pH 7.5), 5% milk powder) for one hour, shaking. <br/>
-<br/>
-<b>Primary antibody incubation:</b><br/>
-A 1:10 dilution of the B1 antibody in TBS + 1% milk was prepared. Membrane was incubated in 50 mL falcon over night at 4°C, rotating.
 ====<p style="font-size:15px; background-color:#66bbff;">Midi-Prep</p>====
@@ Line 2,616: / Line 664: @@
 '''Investigator: Chris W. <br>'''
 <p style="font-size:13px; color:#003399;"> Midi-Prep of:</p><br/>
-pSB1C3_CMV_VP123_453_Z34C =P832 =B656<br/>
+pSB1C3_lITR_CMV_betaglobin_mVenus_hGH_rITR clone1 =P901 =B200<br/>
-pSB1C3_CMV_VP123_453_RGD_HSPG-KO =P833 =B658<br/>
+pSB1C3_lITR_phTERT_betaglobin_mGMK_TK30_hGH_rITR_SDM-PstI clone 2 =P902 =B697<br/>
-pSB1C3_001_RC_IRCK_HSPG-ko_P5tataless_RFC10 clone 1 =P834 =B561<br/>
-pSB1C3_001_RC_IRCK_P5tataless clone 1 =P835 =B516<br/>
-pSB1C3_lITR_CMV_betaglobin_mVenus_hGH_rITR clone1 =P836 =B200<br/>
-pSB1C3_lITR_CMV_betaglobin_mVenus_hGH_rITR clone1 =P837 =B200<br/>
@@ Line 2,628: / Line 674: @@
 {| border="1"
-| plasmid-no. || align="right" |P832|| align="right" |P833|| align="right" |P834|| align="right" |P835|| align="right" |P836|| align="right" |P837
+| plasmid-no. || align="right" |P901|| align="right" |P902
 |-
-| concentration (ng/µl)|| align="right" |3615,8 || align="right" |1504,7 || align="right" |x|| align="right" |x|| align="right" |x|| align="right" |x
+| concentration (ng/µl)|| align="right" |1563,63 || align="right" |1348,26
 |}
 <br>
 <br/>
+====<p style="font-size:15px; background-color:#66bbff;"><b>Continuation of PCR of mGMK and SR39</b></p>====
+<b>Investigator: Jessica</b><br>
-<p style="font-size:13px; color:#003399;"> Comment: P834-837 given to sven</p><br/>
+* vector (P320) and PCR product was digested<br>
-====<p style="font-size:15px; background-color:#66bbFF;"><b>Repetiton: Test digestion of pSB1C3_lITR_phTERT_betaglobin_mGMK_TK30_SDM-PstI_hgH_rITR</b></p>====
-<b>Investigator: Stefan</b><br>
-<p style="color:#66bbff;"><i>Comment</i>: Repetiton of yesterday's test digestion which yielded no clear band at 2000 bp as it was expected. </p>
 {| border="1"
-| align="left" | '''Components''' ||align="left"| <b>P822/P823 / µl</b>
+| align="left" | '''Components''' ||align="left"| <b>P320 / µl</b>||align="left"| <b>PCR product P804 and P860 / µl</b>
 |-
-| align="left" | DNA   ||align="left"| 1,5
+| align="left" | DNA   ||align="left"| 1,5||align="left"| 20
 |-
-| align="left" | Buffer 4 ||align="left"| 1
+| align="left" | Buffer  ||align="left"| 2||align="left"| 3
 |-
-| align="left" | BSA (10x)  ||align="left"| 1
+| align="left" | BSA (10x)  ||align="left"| 2||align="left"| 3
 |-
-| align="left" | SpeI ||align="left"| 0,3
+| align="left" | AgeI ||align="left"| 1 ||align="left"| 1,5
 |-
-| align="left" | XbaI ||align="left"| 0,3
+| align="left" | XbaI ||align="left"| 1 ||align="left"| 1,5
 |-
-| align="left" | H<sub>2</sub>O ||align="left"| 5,9
+| align="left" | H<sub>2</sub>O ||align="left"| 14,5||align="left"| 1
 |-
-| align="left" | '''Total volume''' ||align="left"| <b>10</b>
+| align="left" | '''Total volume''' ||align="left"| <b>20</b>  ||align="left"| <b>30</b>
 |}
+[[Image:Freiburg10 pcr mGMK and SR39.jpg|thumb|center|800px]]
+<br>
+<br>
+'''Ligation'''
+* P320 c= 5,08 ng/µl
+* P804 c= 11,73 ng/µl
+* P860 c= 26,57 ng/µl
+<br>
+* P320 + P804: 4,93µl : 3,07µl
+* P320 + P860: 6,28µl : 1,72µl
+<br>
+'''Transformation with BL21 and Cm'''
+====<p style="font-size:15px; background-color:#66bbff;">Cloning of CMV into pSB1C3_001_VP1, pSB1C3_001_VP2 and pSB1C3_001_VP3</p>====
-Gel:<br>
+'''Investigator: Kerstin, Anna'''
-,45g agarose, 50 ml TAE (0,9%), 3 µl GELRED, 90 Volt, running time ~110 minutes<br>
+<br>
-[[Image:Freiburg10 td131010.jpg|550px]]
-<p style="color:#66bbff;"><i>Comment</i>: This time both samples look well. P822 will be sent for sequencing.</p>
-====<p style="font-size:15px; background-color:#66bbFF;"><b>Sequencing analysis: pSB1C3_leftITR_pCMV_betaglobin_mGMK_TK30_hgH_rITR (P825)</b></p>====
-<b>Investigator: Stefan</b><br>
-For sequencing the following primers were used_
-<ul>
-<li>qPCR primer CMV for (O20)</li>
-<li>GOI primer for (O30)</li>
-<li>seq. primer TK GMK for (O23)</li>
-<li>TK30 SDM Pst for (O191)</li>
-</ul>
-Sequencing comfirmed that the pysical sequence fits our theoretical. Next step will be to remove the remaining PstI restriction site within TK30.
-====<p style="font-size:15px; background-color:#66bbFF;"><b>Repetition of cloning of Rep into Rep78</b></p>====
-<b>Investigator: Kira</b><br/>
-'''Comment:''' The last 2 approaches to digest Rep78 and to clone failed because Rep78 was almost gone after PCR purification and was not detectable on the gel, even if I used the double amount of DNA. Thus, new over night culture was grown in order to perform the experiment again <br />
-Regarding the activity conditions of the enzymes, digestion was performed in 2 steps.  <br />
+'''Plasmids:'''
+*P888: pSB1C3_001_VP3, c =  300,8 ng/µl
+*P890: pSB1C3_001_VP2, c =  298,3 ng/µl
+*P898: pSB1C3_001_VP1, c =  272,7 ng/µl
+*P727: pSB1C3_001_CMV, c =  225,5 ng/µl
+<br>
+'''Digestion:'''
 {| border="1"
-| align="left" | '''Components''' ||align="left"| <b>Rep78</b>
+| <b>components</b>   || align="right" |<b>VP3</b> || align="right" |<b>VP2</b> || align="right" |<b>VP1</b>|| align="right" |<b>CMV</b>
 |-
-| align="left" | DNA   ||align="left"| 3 µl
+| DNA  ||  align="right" |4 ||  align="right" |4||  align="right" |4||  align="right" |8
 |-
-| align="left" | BSA (100x) ||align="left"| 2 µl
+| BSA (10x) ||  align="right" |2 ||  align="right" |2||  align="right" |2||  align="right" |2
 |-
-| align="left" | Buffer no. 3  ||align="left"| 2,0 µl
+| Buffer 4 (10x)||  align="right" |2 ||  align="right" |2 ||  align="right" |2||  align="right" |2
 |-
-| align="left" | Enzyme SwaI  ||align="left"|1,0 µl
+|Enzyme EcoI||  align="right" |1||  align="right" |1 ||  align="right" |1||  align="right" |1
 |-
-| align="left" | H<sub>2</sub>O ||align="left"| 12 µl
+|Enzyme XbaI||  align="right" |1||  align="right" |1 ||  align="right" |1 ||  align="right" |-
 |-
-| align="left" | '''Total volume''' ||align="left"| <b>20</b>
+|Enzyme SpeI||  align="right" |-||  align="right" |- ||  align="right" |-||  align="right" |1
+|-
+|H2O||  align="right" |10||  align="right" |10 ||  align="right" |10  ||  align="right" |10
+|-
+|'''Total '''||  align="right" | 20||  align="right" | 20||  align="right" | 20
 |}
-<br />
+<br>
-incubation @ 25 C for approx. 2 h <br />
+*Digestion: 2h @ 37°C
-Dephosphorylation was performed because of the blunt cleavage. PCR phosphorylation followed in order to get rid off the buffer because Buffer 3 is not appropriate for further digestion.
+<br>
+'''Gel:'''
+*1% agarose gel, 1 µl Gelred, run for 45 min
+[[Image:Freiburg10_Cloning of CMV into pSB1C3_001_VP1-3.jpg|400px]]
+<br>
+'''Gel extraction'''
 {| border="1"
-| align="left" | '''Components''' ||align="left"| <b>Rep78</b>
+|sample name || align="right" |<b>VP3</b>|| align="right" |<b>VP2</b>|| align="right" |<b>VP1</b>|| align="right" |<b>CMV</b>
 |-
-| align="left" | DNA   ||align="left"| 30 µl
+|nanodrop concentrations || align="right" |44,36|| align="right" |32,33|| align="right" |22,73|| align="right" |18,5
 |-
-| align="left" | BSA (100x) ||align="left"| 0 µl
+|expected fragment size|| align="right" |4100|| align="right" |4000|| align="right" |3700|| align="right" |650
 |-
-| align="left" | Buffer no. 2  ||align="left"| 4,5 µl
+|}
+<br>
+'''Ligation:'''
+{| border="1"
+|ligation name || align="right" |<b>VP3 + CMV</b>|| align="right" |<b>VP2 + CMV</b>|| align="right" |<b>VP1 + CMV</b>
 |-
-| align="left" | Enzyme HindIII  ||align="left"|1,0 µl
+|volume of vector || align="right" |5,7|| align="right" |4,3|| align="right" |5
 |-
-| align="left" | H<sub>2</sub>O ||align="left"| 9,5 µl
+|volume of insert|| align="right" |3,3|| align="right" |3,7|| align="right" |3
+|-
+|T4 ligase buffer (10x)|| align="right" |1|| align="right" |1|| align="right" |1
+|-
+|T4 ligase || align="right" |1|| align="right" |1|| align="right" |1
 |-
-| align="left" | '''Total volume''' ||align="left"| <b>45</b>
 |}
-<br />
+*Ligation @ RT for 30 min
-incubation for 2h at 37C<br />
+'''Trafo:'''
+<br>
-[[Image: Rep78 2010_10_13.jpg]]
+Was done following the standard protocol using BL21 cells.
-Ligation with T4 ligase <br />
-v(insert)=4,4 ul<br />
+<br>
-v(vector)= 3,6 ul<br />
-T4 buffer= 1 ul<br />
-T4 ligase= 1 ul<br />
-incubation at RT for 45 min<br />
+===<p style="font-size:17px; background-color:#00dd77;">155. labday 20.10.2010</p>===
-Transformation according to the standard protocol with BL21 <br />
 ====<p style="font-size:15px; background-color:#66bbff;">Midi-Prep</p>====
@@ Line 2,742: / Line 798: @@
 '''Investigator: Chris W. <br>'''
 <p style="font-size:13px; color:#003399;"> Midi-Prep of:</p><br/>
-pSB1C3_CMV_VP123_capins =P862 =B474<br/>
+pSB1C3_lITR_pTERT_ßglobin_CD_hGH_rITR clone 1 =P903 =B702<br/>
-pSB1C3_P40_VP123 =P863 =B651<br/>
+pSB1C3_lITR_CMV_ßglobin_CD_hGH_rITR clone 1 =P904 =B712<br/>
@@ Line 2,751: / Line 808: @@
 {| border="1"
-| plasmid-no. || align="right" |P862|| align="right" |P863
+| plasmid-no. || align="right" |P903|| align="right" |P904
 |-
-| concentration (ng/µl)|| align="right" |3310,5 || align="right" |2209,2
+| concentration (ng/µl)|| align="right" |832,63 || align="right" |1174,49
 |}
 <br>
 <br/>
-<html>
+===<p style="font-size:17px; background-color:#00dd77;">156. labday 21.10.2010</p>===
-</div>
+====<p style="font-size:15px; background-color:#66bbff;">ÄKTA Chromatography and Ultrafiltration of virus particles </p>====
-</html>
-===<p style="font-size:17px; background-color:#00dd77;">149. labday 14.10.2010</p>===
-====<p style="font-size:15px; background-color:#66bbFF;"><b>Continuation of Western Blot of unmodified AAV2 capsids</b></p>====
-<b>Investigator: Hanna</b>
-<br/>
-<b>Comment:</b> Yesterday, SDS PAGE, Western Blot and primary B1 mouse antibody incubation was performed. <br/>
-Today, secondary antibody incubation and ECL analysis will be done. <br/>
-<br/>
-* Membrane was washed 3 x 7 minutes in TBST (0.2% Tween).
-* A 1:5000 dilution of secondary goat anti-mouse HRP (sc2005) coupled antibody in TBS was prepared.
-* Membrane was incubated in secondary antiobody for 1 hour.
-* Membrane was washed 3 x 10 minutes.
-* Pierce ECL Western Blotting Substrate was prepared: 350 µL Luminol Enhancer Solution + 350 µL Peroxidase Solution
-* Analysis of the membrane revealed that the samples didn't run well. Much unspecific antibody binding was detectable.
-<br/>
-Because of these dissatisfying results SDS PAGE will be repeated. Instead of performing Western Blot, gel wioll be stained by Coomassie in order to optimize SDS-PAGE conditions. <br/>
-====<p style="font-size:15px; background-color:#66bbFF;"><b>Cloning hGH_rITR into (form P728) into pSB1C3_lITR_phTERT_betaglobin_mGMK_TK30_SDM-PstI (P822) </b></p>====
-Investigator Patrick
-Digestions, 1 h 40 minutes, 37 °C:
-*P728: 1 µl XbaI, 1 µl PstI, 2 µl BSA, 2 µl Buffer 4, 6 µl DNA, 8 µl H2O
-*P822: 1 µl SpeI, 1 µl PstI, 2 µl BSA, 2 µl Buffer 4, 6 µl DNA, 8 µl H2O
-Expected results for the 1% agarose gel:
-*P728: about 2100 and 650 bp
-*P822: about about 4900 and 10 bp (wont be visible on the gel)
-<br>
-[[Image:Freiburg10_1014pat.jpg]]
-<br>
-<br>
-As usual the PSB1C3 bands indicate a higher bp amount than they actually got.
-<br>
-The gelextraction ...
-*P728: 9 ng/µl
-*P822: 67,5 ng/µl
-<br>
-... and following ligation (2 µl Insert, 6 µl vector, 1 µl T4 DNA Ligase, 1 µl T4 DNA Ligase Buffer, 40 minutes, RT) were performed according to the standard protocol. After the transformation (with BL21) the cells were plated and put into the 37°C room.
-====<p style="font-size:15px; background-color:#66bbFF;"><b>Cloning of the ViralBricks 453-empty and 587 empty </b></p>====
-These ViralBricks were not ordered as hybridized oligos, but can be cloned from the Cap-Gene-Synthesis into the ViralBrick MCS.
+'''Investigator: Hanna <br>'''
+<b>ÄKTA chromatography</b> with VP1up_NLS_mVenus_VP2/3 containing virus particles was conducted. Fraction 5 - 10 delivered highest protein concentrations. <br/>
 {| border="1"
-| components
+|<b>Sample</b> || align="right" |<b>A(260 nm)</b> || align="right" |<b>A(280 nm)</b>|| align="right" |<b>A(515 nm) (YFP)</b>
-|volume of p320 (ViralBrick-BLA)  /µl
-|volume of p320 (ViralBrick-BLA) /µl
-|volume of p668 (pSB1C3_RepCap_IRCK) /µl
-|volume of p668 (pSB1C3_RepCap_IRCK) /µl
 |-
-| DNA
+| 5  ||  align="right" |0.032 ||  align="right" |0.027||  align="right" |0.003
-|2µl
-|2µl
-|7µl
-|7µl
 |-
-| BSA (100x)
+| 6 ||  align="right" |0.019 ||  align="right" |0.019||  align="right" |0.003
-|2.5µl
-|2.5µl
-|2.5µl
-|2.5µl
 |-
-| Buffer 4 (10x)
+| 7||  align="right" |0.075 ||  align="right" |0.09 ||  align="right" |0.01
-|2.5µl
-|2.5µl
-|2.5µl
-|2.5µl
 |-
-|SspI
+|8||  align="right" |0.054||  align="right" |0.075 ||  align="right" |0.007
-|1µl
-|0
-|1µl
-|0
 |-
-|SalI
+|10||  align="right" |-0.008||  align="right" |-0.008 ||  align="right" |0.005
-|1µl
+|}
-|0
-|1µl
+<br/>
-|0
+A further attempt was conducted which included digestion with Benzonase (1 hour) prior to ÄKTA chromatography. Following protein concentrations were obtained: <br/>
+{| border="1"
+|<b>Sample</b> || align="right" |<b>A(260 nm)</b> || align="right" |<b>A(280 nm)</b>|| align="right" |<b>A(515 nm) (YFP)</b>
 |-
-|BamHI
+| 5  ||  align="right" |0.005||  align="right" |0.007||  align="right" |0.003
-|0
-|1µl
-|0
-|1µl
 |-
-|PvuII
+| 6 ||  align="right" |0.047||  align="right" |0.041||  align="right" |0.006
-|0
-|1µl
-|0
-|1µl
 |-
-|H<sub>2</sub>O
+| 7||  align="right" |0.151||  align="right" |0.153||  align="right" |0.01
-|16µl
-|16µl
-|11µl
-|11µl
 |-
-|'''Total volume
+|8||  align="right" |0.172||  align="right" |0.2||  align="right" |0.009
-|25µl
-|25µl
-|25µl
-|25µl
 |-
+|9||  align="right" |0.098||  align="right" |0.128||  align="right" |0.009
+|-
+|10||  align="right" |0.053||  align="right" |0.074||  align="right" |0.005
 |}
-<li> Incubation: 4h<br><br><br>
-{| border="1"
-|
-!Sample Marker
-!Sample ViralBrick_BLA 453 cut
-!Sample ViralBrick_BLA 587 cut
-!Sample pSB1C3_001_RepCap_IRCK 453 cut
-!Sample pSB1C3_001_RepCap_IRCK 587 cut
-|-
-!Lane
-|7µl
-|30µl
-|30µl
-|30µl
-|30µl
-|-
-|}<br>
-[[Image:Freiburg10_ViralBrick 453 empty and 587 empty.jpg|thumb|center|600px]]
 <br/>
-====<p style="font-size:15px; background-color:#66bbFF;"><b>SDS PAGE of viral capsids</b></p>====
-<b>Investigator: Hanna</b>
-<br/>
-<b>Comment:</b> In order to optimize SDS PAGE with the viral capsid proteins, I didn't perform Western Blot, but Coomassie staining.
 <br/>
+<b>Ultrafiltration</b> <br/>
+Ultrafiltration of CFP_MiddleLinker_VP2/3 containing virus particles and 587-BAP virus particles were concentrated via Vivaspin-Ultrafiltration: <br/>
+*20 mL virus containing cell culture supernatant was added to GE Vivaspin 20 filter and centrifuged with 4000 g at 15°C until 750 - 1000 µL was left-
+* 5 mL Bis-Trus buffer (pH 6) was added and centrifuged again with 4000 g at 15°C (washing).
+* This step was repeated 3 more times.
+* Membrane was carefully resuspended and cleared. Suspension was transfered to low-binding eppi and centrifuged with 10000 g for 10 minutes at 15°C.
+* Supernatant was transfered to new low-binding eppi and again centrifuged with 10000 g for 10 minutes at 15°C.
+* Supernatant was transfered to new low-binding eppi and stored at 4°C over night. <b>To do:</b> ÄKTA chromatography. <br/>
-<b>1.: Preparation of 10% polyacrylamide gel: </b><br/>
-Following standard protocol.<br/>
-<b>2.: Preparation of virus samples: </b><br/>
+====<p style="font-size:15px; background-color:#66bbff;">MTT Assay: Testing Superconstructs </p>====
-Two different virus stocks with unmodified capsid were taken. 2 samples were prepared of each: One sample was untreated, the other one was centrifuged in order to sediment cell fragments. <br/>
+'''Investigator: Anissa, Kerstin <br>'''
-µL Laemmli buffer (5x) was added to 40 µL virus sample and incubated at 95°C for 5 minutes. <br/>
-<b>3.: SDS PAGE </b><br/>
+[[Image:Freiburg10 Transductionplan1 21.10.2010.jpg|700px]]
-Loading plan: <br/>
+[[Image:Freiburg10 Transductionplan2 21.10.2010.jpg|700px]]
-{| border="1"
+[[Image:Freiburg10 Transductionplan3 21.10.2010.jpg|700px]]
-| align="left" |'''10 µL PageRuler Prestained Protein Ladder'''||align="left"|<b>10 µL Sample 1 (not centrifuged)</b>||align="left"|<b>20 µL Sample 1 (not centrifuged)</b>||align="left"|<b>10 µL Sample 1 (centrifuged)</b>||align="left"|<b>20 µL Sample 1 (centrifuged)</b>||align="left"|<b>10 µL Sample 2 (not centrifuged)</b>||align="left"|<b>20 µL Sample 2 (not centrifuged)</b>||align="left"|<b>10 µL Sample 2 (centrifuged)</b>||align="left"|<b>20 µL Sample 2 (centrifuged)</b>
+[[Image:Freiburg10 Transductionplan4 21.10.2010.jpg|700px]]
-|}
+[[Image:Freiburg10 Transductionplan5 21.10.2010.jpg|700px]]
-<br/>
-Gel was run 1.5 hours at 10 mA.
-<br/>
-<b>4.: Coomassie staining </b><br/>
+===<p style="font-size:17px; background-color:#00dd77;">157. labday 22.10.2010</p>===
-Gel was put into coomassie bath, heated in microwave and incubated for 1 hour, shaking. <br/>
+====<p style="font-size:15px; background-color:#66bbff;">SDS PAGE and Coomassie staining</p>====
-In order to remove background coomassie staining, gel was incubated in acetic acid (20%) for 2 hours, shaking. <br/>
-<br/>
-[[Image:Freiburg10 sdspage.png|400px|thumb|center|SDS PAGE of AAV2 stock - unmodified capsids]]<br/>
-<br/>
-Results revealed that samples ran well (vertically :) ) - 20 µL seems to be too much. Interestingly centrifuging the virus stocks in order to remove cell debris didn't lead to better results. <br/>
-<br/>
-<b>To do:</b> Perform Western Blot again - with 10 µL Virus stock, 0.5% instead of just 0.2% TBS-milk and longer washing steps. <br/>
-====<p style="font-size:15px; background-color:#66bbFF;"><b>Sequence analysis of super constructs</b></p>====
+'''Investigator: Hanna <br>'''
-<b>Investigator: Hanna</b>
 <br/>
-<b>Comment:</b> We assembled targeting super constructs which specifically bind the EGF receptor with VP2 N-terminal fused Z<sub>EGFR:1907</sub> Affibody and can either be purified via a loop inserted His-Tag or visualized via Streptavidin coupled fluorescent proteins. <br/>
+Prior to performing Western Blot we decided to investigate running behaviour of different samples. <br/>
-Further on these loop motifs were vloned to the "mVenus VP1 insertion". <br/>
+* 1. Cell debris (control)
-<br/>
+* 2. Cell debris containing virus particles
-[[Image:Freiburg10 Super BAP.png|450px|thumb|left]]
+* 3. Concentrated virus stock (containing CFP_MiddleLinker_VP2/3)
-[[Image:Freiburg10 Super His.png|450px|thumb|right]]<br/>
+* 4. ÄKTA purified virus stock: Fraction 6
-[[Image:Freiburg10 VP1 BAP.png|450px|thumb|left]]
+* 5. ÄKTA purified virus stock: Fraction 7
-[[Image:Freiburg10 VP1 His.png|450px|thumb|right]]
+* 6. Benzonase treated, ÄKTA purified virus stock: Fraction 7
-<br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/>
+* 7. Benzonase treated, ÄKTA purified virus stock: Fraction 8
-Sequencing results showed that insertion of these constructs into pSB1C3_CMV worked! <br/>
-<br/>
-<b>Next steps:</b> Put sample sinto BioBrick Box. <br/>
-Midi-Prep + Transfection. <br/>
-====<p style="font-size:15px; background-color:#66bbFF;"><b>Transfection with pCMV, p40, pAAV_RC</b></p>====
-<b>Investigator: Kira</b>
 <br/>
-'''Comment''': Transfection was performed in order to evaluate the promoter activities.
+µL Laemmli buffer was added to 20 µL sample. Samples were incubated at 95°C for 8 minutes and loaded onto a SDS gel (10 %). SDS PAGE was performed at 90 V (collection gel) resp. 120 V (separation gel). <br/>
+Gel was put into Coomassie dye, heated for 30 seconds in microwave and incubated for 1 hours shaking. <br/>
-Yesterday, the cells were seeded in the 6 well plate by Sven. Transformation was performed according to the manufacturer protocol (Qiagen).
+Gel was decolorized in acetic acid (20%). <br/>
+Loading plan:
-====<p style="font-size:15px; background-color:#66bbFF;"><b>Trafo evaluation of Rep78</b></p>====
+{| border="1"
-<b>Investigator: Kira</b>
+|<b>Marker</b> || align="right" |<b>Concentrated Stock</b> || align="right" |<b>ÄKTA 6</b>|| align="right" |<b>ÄKTA 7</b>|| align="right"|<b>Benzonase/ÄKTA 7</b>|| align="right" |<b>Benzonase/ÄKTA 8</b>|| align="right"|<b> - - - </b>|| align="right" |<b> - - - </b>|| align="right"|<b>Cell debris</b>|| align="right" |<b>Cell debris + Virus</b>
+|}
 <br/>
-Transformation was successful. 2 clones will be picked for mini prep tomorrow.
+[[Image:Freiburg10 SDSVersuch2.jpg|500px|center]]
-====<p style="font-size:15px; background-color:#66bbFF;"><b>Picking clones</b></p>====
-<b>Investigator: Kira</b>
 <br/>
+Gel picture shows that concentration works :) <br/>
+In addition to that one can see that the BSA bands disappear after ÄKTA chromatography. <br/>
+<b>Next step:</b> Western Blot of ÄKTA purified virus stocks. <br/>
-Rep78 and Rep52 were picked (2 clones from each plate), in order to continue with mini prep and PCR amplification.
+===<p style="font-size:17px; background-color:#00dd77;">158. labday 23.10.2010</p>===
-===<p style="font-size:17px; background-color:#00dd77;">150. labday 15.10.2010</p>===
+===<p style="font-size:17px; background-color:#00dd77;">159. labday 24.10.2010</p>===
-====<p style="font-size:15px; background-color:#66bbff;">Cloning CFP (from P666: PSB1C3_CFP) into pSB1C3_leftITR_CMV_beta-globin (P729)</p>====
-Investigator Patrick
 ====<p style="font-size:15px; background-color:#66bbff;">Midi-Prep</p>====
@@ Line 2,968: / Line 909: @@
 '''Investigator: Chris W. <br>'''
 <p style="font-size:13px; color:#003399;"> Midi-Prep of:</p><br/>
-pSB1C3_001_RC_IRCK_P5tataless clone 1 =P866 =B516<br/>
+pSB1C3_001_RC_IRCK_VP2-ko_HSPG-ko_P5tataless cl1 =P966 =B523<br/>
-pSB1C3_001_CMV_VP123_587-KO_Z34C_spacer clone2 =P867 =B526<br/>
-pSB1C3_001_CMV_VP123_587-KO_Z34C clone2 =P868 =B529<br/>
-pSB1C3_CMV_Zegfr:1907_MiddleLinker_VP2/3_587-KO_BAP clone 1 =P869 =B680<br/>
-pSB1C3_CMV_Zegfr:1907_MiddleLinker_VP2/3_587-KO_6xHis clone 1 =P870 =B200<br/>
 <br/>
-The Midi-Preps were performed according to the standard protocol yielding the following concentrations:
+The Midi-Prep were performed according to the standard protocol yielding the following concentration:
 {| border="1"
-| plasmid-no. || align="right" |P866|| align="right" |P867|| align="right" |P868|| align="right" |P869|| align="right" |P870
+| plasmid-no. || align="right" |P966
 |-
-| concentration (ng/µl)|| align="right" |899,80 || align="right" |954,46 || align="right" |406,97|| align="right" |1642,76|| align="right" |1585,12
+| concentration (ng/µl)|| align="right" |310,69
 |}
 <br>
 <br/>
-====<p style="font-size:15px; background-color:#66bbff;">mini prep of several constructs</p>====
-Investigator:  Kira <br/>
-c(rep52_1)=299,04 ng/ul<br/>
+===<p style="font-size:17px; background-color:#00dd77;">160. labday 25.10.2010</p>===
-c(rep52_2)=290,07 ng/ul<br/>
-c(rep78_1)=142,32 ng/ul<br/>
-c(rep78_2)=175,36 ng/ul<br/>
-===<p style="font-size:17px; background-color:#00dd77;">151. labday 16.10.2010</p>===
+===<p style="font-size:17px; background-color:#00dd77;">161. labday 26.10.2010</p>===
-===<p style="font-size:17px; background-color:#00dd77;">152. labday 17.10.2010</p>===
+===<p style="font-size:17px; background-color:#00dd77;">162. labday 27.10.2010</p>===
-===<p style="font-size:17px; background-color:#00dd77;">153. labday 18.10.2010</p>===
+===<p style="font-size:17px; background-color:#00dd77;">163. labday 28.10.2010</p>===
-===<p style="font-size:17px; background-color:#00dd77;">154. labday 19.10.2010</p>===
+===<p style="font-size:17px; background-color:#00dd77;">164. labday 29.10.2010</p>===
-===<p style="font-size:17px; background-color:#00dd77;">155. labday 20.10.2010</p>===
+===<p style="font-size:17px; background-color:#00dd77;">165. labday 30.10.2010</p>===
+===<p style="font-size:17px; background-color:#00dd77;">166. labday 31.10.2010</p>===
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+<center>[https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/November '''=> Go to Labjournal November (labday 167 - 170 )''']</center><br>
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Team:Freiburg Bioware/NoteBook/Labjournal/October2

From 2010.igem.org

Latest revision as of 21:33, 27 October 2010

Contents

150. labday 15.10.2010

Cloning CFP (from P666: PSB1C3_CFP) into pSB1C3_leftITR_CMV_beta-globin (P729)

Midi-Prep

mini prep of several constructs

Cell culture

Mini-Prep and test digestion of pSB1C3_CD_SDM-PstI_hGH_rITR

151. labday 16.10.2010

Biobrick assembly: pSB1C3_lITR_CMV_ß-globin_CD_hGH_rITR and pSB1C3_lITR_phTERT_ß-globin_CD_hGH_rITR

Mini-prep of mutual pSB1C3_lITR_phTERT_betaglobin_mGMK_TK30_hGH_rITR_SDM-PstI

Biobrick assembly of Rep78 and Rep52

RNA harvesting

152. labday 17.10.2010

Test digestion of pSB1C3_lITR_CMV_ß-globin_CFP

Cloning of hGH_rITR into pSB1C3_lITR_CMV_betaglobin_CFP

RT-PCR

153. labday 18.10.2010

quantitative real-time PCR for detection of virus titer

Test-digestion of pSB1C3_lITR_phTERT_betaglobin_mGMK_TK30_hGH_rITR_SDM-PstI clone 1, 2 and 3 (P877, 878 and 879)

MTT Assay

Mini-Prep and test digestion of several constructs

PCR of mGMK and SR39

154. labday 19.10.2010

Midi-Prep

Continuation of PCR of mGMK and SR39

Cloning of CMV into pSB1C3_001_VP1, pSB1C3_001_VP2 and pSB1C3_001_VP3

155. labday 20.10.2010

Midi-Prep

156. labday 21.10.2010

ÄKTA Chromatography and Ultrafiltration of virus particles

MTT Assay: Testing Superconstructs

157. labday 22.10.2010

SDS PAGE and Coomassie staining

158. labday 23.10.2010

159. labday 24.10.2010

Midi-Prep

160. labday 25.10.2010

161. labday 26.10.2010

162. labday 27.10.2010

163. labday 28.10.2010

164. labday 29.10.2010

165. labday 30.10.2010

166. labday 31.10.2010