Team:Freiburg Bioware/NoteBook/Labjournal/October2

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146. labday 11.10.2010

Colony PCR of Cloning VP2 Fusion and Super constructs into pSB1C3

Investigator: Achim, Hanna

Comment: 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.
Two clones were picked from each plat. In addition to that a positive (pAAV_RC) and a negative control (pSB1C3_lITR) was prepared.
Used primer: 4200 rev and Cap3500 for. Expected fragment size: 885 bp.
PCR was performed following the standard protocol.

All samples match the positive control!
To do: Mini-Prep and sequencing.

Preparation of SDS-PAGE gel (10%)

Investigator: Hanna

Comment: In order to perform a Western Blot of different virus capsids (with and without capsid-motifs), 2 10% SDS polyacrylamid gels were prepared.

Resolving gel: 15 mL

• H₂O: 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

Stacking gel (5%): 5 mL

• H₂O: 3.4 mL
• Acryl-bisacrylamide mix (30%): 0.83 mL
• Tris (1.5 M, pH 6.8): 0.63 mL
• SDS (10%): 0.05 mL
• Ammonium persulfate (10%): 0.05 mL
• TEMED: 0.005 mL

Colony PCR of p40_VP123_capins

Investigator: Bea

Comment: 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.

Protocol:

• Primer used: O162
• Primer used: O38

The PCR products were loaded on a 1% agarose gel. The results can be seen above in the gel picture:

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.

Cloning of lITR_CMV_betaglobin and lITR_phTERT_betaglobin into pSB1C3_CD

Investigator: Stefan

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.

Vector name:
pSB1C3_CD clone 1
pSB1C3_CD clone 2

Insert name:
pSB1C3_lITR_CMV_beta-globin (P729)
pSB1C3_lITR_phTERT_beta-globin (P730)

Digestion:

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

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.

Gel extraction:
Was performed according to protocol.

T4 Ligation:

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

Seeding HT1080 and A431 for testing different concentrations of ganciclovir by MTT-Assay

Investigator: Kerstin, Anissa

• Seeding of 4x 96-well plates:

• Transduction plan (12.10.2010):

FACS-Analysis

Investigator: Kerstin

• Results Transduction

Preparation of the ELISA

Investigator: Volker

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:

147. labday 12.10.2010

Concentration of virus stock for Western Blot analysis

Investigator: Hanna

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.
For this purpose 8 mL virus stock were concentrated via amicon filtration.

• 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.
• 2 x 20 µL protein solution was taken and mixed with Laemmli buffer and stored over night @ 4°C.

To do: SDS-PAGE and Western Blot tomorrow.
Perfomance of another try by harvesting viruses of the cell pellet with RIPA buffer & freeze/ thaw.
Western Blot of unmodified, and modified capsids (N-terminal fusion and VP1 insertion with CFP ref. mVenus) --> size shift should be detectable.

Mini-Prep and test digestion of several constructs

Investigator: Stefan

Glycerol stocks were prepared:

• B651 = pSB1C3_P40_VP123
• B652 = pSB1C3_litr_phTERT_ß-globin_mGMK_TK30_pSTI_SDM_hgH_rITR
• B653 = pSB1C3_litr_phTERT_ß-globin_mGMK_TK30_pSTI_SDM_hgH_rITR
• B654 = pSB1C3_Iitr_CMV_ß-globin_mGMK_TK30_hgH_rITR
• B655 = pSB1C3_Iitr_CMV_ß-globin_mGMK_TK30_hgH_rITR
• B656 = pSB1C3_CMV_VP123_453_Z34C_HSPG-KO
• B657 = pSB1C3_CMV_VP123_453_Z34C_HSPG-KO
• B658 = pSB1C3_CMV_VP123_453_RGD_HSPG-KO
• B659 = pSB1C3_CMV_VP123_453_RGD_HSPG-KO
• B660 = pSB1C3_litr_phTERT_ß-globin_cfp_hgH-ritr
• B661 = pSB1C3_litr_phTERT_ß-globin_cfp_hgH-ritr

Mini-Prep was performed according to standard protocol:

• P821 = pSB1C3_P40_VP123 c = 327,4 ng/µl
• P822 = pSB1C3_litr_phTERT_ß-globin_mGMK_TK30_pSTI_SDM_hgH_rITR c = 342,9 ng/µl
• P823 = pSB1C3_litr_phTERT_ß-globin_mGMK_TK30_pSTI_SDM_hgH_rITR c = 324,0 ng/µl
• P824 = pSB1C3_Iitr_CMV_ß-globin_mGMK_TK30_hgH_rITR c = 81,6 ng/µl
• P825 = pSB1C3_Iitr_CMV_ß-globin_mGMK_TK30_hgH_rITR c = 148,4 ng/µl
• P826 = pSB1C3_CMV_VP123_453_Z34C_HSPG-KO c = 105,8 ng/µl
• P827 = pSB1C3_CMV_VP123_453_Z34C_HSPG-KO c = 121,1 ng/µl
• P828 = pSB1C3_CMV_VP123_453_RGD_HSPG-KO c = 188,4 ng/µl
• P829 = pSB1C3_CMV_VP123_453_RGD_HSPG-KO c = 140,4 ng/µl
• P830 = pSB1C3_litr_phTERT_ß-globin_cfp_hgH-ritr c = 197,2 ng/µl
• P831 = pSB1C3_litr_phTERT_ß-globin_cfp_hgH-ritr c = 207,3 ng/µl

Test digestion:

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

Comment: .

148. labday 13.10.2010

149. labday 14.10.2010

150. labday 15.10.2010

151. labday 16.10.2010

152. labday 17.10.2010

153. labday 18.10.2010

154. labday 19.10.2010

155. labday 20.10.2010