Team:Freiburg Bioware/NoteBook/Labjournal/October2

From 2010.igem.org

(Difference between revisions)
(SDS PAGE and Coomassie staining)
(SDS PAGE and Coomassie staining)
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* Supernatant was transfered to new low-binding eppi and stored at 4°C over night. <b>To do:</b> ÄKTA chromatography. <br/>  
* Supernatant was transfered to new low-binding eppi and stored at 4°C over night. <b>To do:</b> ÄKTA chromatography. <br/>  
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====<p style="font-size:15px; background-color:#66bbff;">SDS PAGE and Coomassie staining</p>====
 
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'''Investigator: Hanna <br>'''
 
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<br/>
 
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Prior to performing Western Blot we decided to investigate running behaviour of different samples. <br/>
 
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* 1. Cell debris (control)
 
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* 2. Cell debris containing virus particles
 
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* 3. Concentrated virus stock (containing CFP_MiddleLinker_VP2/3)
 
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* 4. ÄKTA purified virus stock: Fraction 6
 
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* 5. ÄKTA purified virus stock: Fraction 7
 
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* 6. Benzonase treated, ÄKTA purified virus stock: Fraction 7
 
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* 7. Benzonase treated, ÄKTA purified virus stock: Fraction 8
 
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<br/>
 
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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). <br/>
 
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Gel was put into Coomassie dye, heated for 30 seconds in microwave and incubated for 1 hours shaking. <br/>
 
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Gel was decolorized in acetic acid (20%). <br/>
 
===<p style="font-size:17px; background-color:#00dd77;">157. labday 22.10.2010</p>===
===<p style="font-size:17px; background-color:#00dd77;">157. labday 22.10.2010</p>===

Revision as of 19:03, 22 October 2010

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Contents

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


Freiburg10 1015pat fertig.jpg



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. P866P867P868P869P870
concentration (ng/µl)899,80 954,46 406,971642,761585,12



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

Freiburg10 td151010.jpg

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 1414
BSA (10x) 2 22
Buffer 4 (10x)2 2 2
Enzyme EcoI11 1
Enzyme XbaI-- 1
Enzyme SpeI11 -
H2O8- -
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 + VI2 + V
volume of vector 4.694.23
volume of insert3.313.77
T4 ligase buffer (10x)11
T4 ligase 11
  • 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

Freiburg10 1016pat test.jpg


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


CyclesTemperatureTime
98°C30 sec
10x98°C15 sec
63°C25 sec
72°C32 sec
20x98°C15 sec
66°C25 sec
72°C32 sec
1x72°C5 min
Hold 4°C


1% agarose gel
Freiburg10 Rep78&Rep52 PCR.jpg

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

Freiburg10 digestion pSB1C31 16.10.jpg

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 NgoMIV0,30,3
Enzyme AgeI0,30,3
H2O5,45,4
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 PstI11
Enzyme XbaI-1
Enzyme SpeI1-
H2O114
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



Freiburg10 171010.jpg

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 insert5,32
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
Freiburg10 1019pat test1bfertig.jpg
PstI or BstEII seems to work not properly

MTT Assay

Investigator Kerstin, Anissa

Freiburg10 transductionplan 18.10..jpg

Freiburg10 transductionplan1 18.10..jpg
Freiburg10 transductionplan2 18.10..jpg

Results:

Freiburg10 Results of MTTAssay 18 10 10 2day 1pic.jpg
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

Freiburg10 test digestion 886-899.jpg

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_for2,5
Primer_rev2,5
DNA template1
H2O32,5
Total volume 50


PCR Program:

CyclesTemperatureTime
98°C60 sec
98°C15 sec
8x52°C25 sec
72°C25 sec
98°C15 sec
17x67°C25 sec
72°C25 sec
1x72°C5 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. P901P902
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
Freiburg10 pcr mGMK and SR39.jpg



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 VP1CMV
DNA 4 448
BSA (10x) 2 222
Buffer 4 (10x)2 2 22
Enzyme EcoI11 11
Enzyme XbaI11 1 -
Enzyme SpeI-- -1
H2O1010 10 10
Total 20 20 20


  • Digestion: 2h @ 37°C


Gel:

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

Freiburg10 Cloning of CMV into pSB1C3 001 VP1-3.jpg

Gel extraction

sample name VP3VP2VP1CMV
nanodrop concentrations 44,3632,3322,7318,5
expected fragment size410040003700650


Ligation:

ligation name VP3 + CMVVP2 + CMVVP1 + CMV
volume of vector 5,74,35
volume of insert3,33,73
T4 ligase buffer (10x)111
T4 ligase 111
  • 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. P903P904
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 0.0270.003
6 0.019 0.0190.003
70.075 0.09 0.01
80.0540.075 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 0.0050.0070.003
6 0.0470.0410.006
70.1510.1530.01
80.1720.20.009
90.0980.1280.009
100.0530.0740.005



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

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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