Team:Freiburg Bioware/NoteBook/Labjournal/October2

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<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/September">September part 1 (labday 107 - 123)</a></li>
<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/September">September part 1 (labday 107 - 123)</a></li>
<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/September2">September part 2 (labday 124 - 135)</a></li>
<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/September2">September part 2 (labday 124 - 135)</a></li>
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<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October">October part 1 (labday 136 - 145 )</a></li>
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<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October">October part 1 (labday 136 - 149 )</a></li>
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<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October2">October part 2 (labday 146 - 155 )</a></li>
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<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October2">October part 2 (labday 150 - 166 )</a></li>
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<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October3">October part 3 (labday 156 - 166 )</a></li>
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<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/November">November  (labday 167 - 170 )</a></li>
<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/November">November  (labday 167 - 170 )</a></li>
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<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/Cellculture">Cellculture </a></li>
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<li><a href="http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/Cellculture">Cellculture</a></li>
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Latest revision as of 21:33, 27 October 2010

=> Back to Notebook overview



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.


MTT Assay: Testing Superconstructs

Investigator: Anissa, Kerstin

Freiburg10 Transductionplan1 21.10.2010.jpg Freiburg10 Transductionplan2 21.10.2010.jpg Freiburg10 Transductionplan3 21.10.2010.jpg Freiburg10 Transductionplan4 21.10.2010.jpg Freiburg10 Transductionplan5 21.10.2010.jpg

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 6ÄKTA 7Benzonase/ÄKTA 7Benzonase/ÄKTA 8 - - - - - - Cell debrisCell debris + Virus


Freiburg10 SDSVersuch2.jpg


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

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162. labday 27.10.2010

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166. labday 31.10.2010

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