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Contents 1 150. labday 15.10.2010 1.1 Cloning CFP (from P666: PSB1C3_CFP) into pSB1C3_leftITR_CMV_beta-globin (P729) 1.2 Midi-Prep 1.3 mini prep of several constructs 1.4 Cell culture 1.5 Mini-Prep and test digestion of pSB1C3_CD_SDM-PstI_hGH_rITR 2 151. labday 16.10.2010 2.1 Biobrick assembly: pSB1C3_lITR_CMV_ß-globin_CD_hGH_rITR and pSB1C3_lITR_phTERT_ß-globin_CD_hGH_rITR 2.2 Mini-prep of mutual pSB1C3_lITR_phTERT_betaglobin_mGMK_TK30_hGH_rITR_SDM-PstI 2.3 Biobrick assembly of Rep78 and Rep52 2.4 RNA harvesting 3 152. labday 17.10.2010 3.1 Test digestion of pSB1C3_lITR_CMV_ß-globin_CFP 3.2 Cloning of hGH_rITR into pSB1C3_lITR_CMV_betaglobin_CFP 3.3 RT-PCR 4 153. labday 18.10.2010 4.1 quantitative real-time PCR for detection of virus titer 4.2 Test-digestion of pSB1C3_lITR_phTERT_betaglobin_mGMK_TK30_hGH_rITR_SDM-PstI clone 1, 2 and 3 (P877, 878 and 879) 4.3 MTT Assay 4.4 Mini-Prep and test digestion of several constructs 4.5 PCR of mGMK and SR39 5 154. labday 19.10.2010 5.1 Midi-Prep 5.2 Continuation of PCR of mGMK and SR39 5.3 Cloning of CMV into pSB1C3_001_VP1, pSB1C3_001_VP2 and pSB1C3_001_VP3 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 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 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

• 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

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:

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.
  

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:

158. labday 23.10.2010

159. labday 24.10.2010

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

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