Team:Heidelberg/Notebook/Homology Based/October
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* AAVs from the first selection round on primary hepatocytes were harvested using freeze-thaw cycles as before (see previous days), and the supernatants were kept at -20 C for the second selection round (primary hepatocytes are received every Monday). | * AAVs from the first selection round on primary hepatocytes were harvested using freeze-thaw cycles as before (see previous days), and the supernatants were kept at -20 C for the second selection round (primary hepatocytes are received every Monday). | ||
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+ | ==16/10/2010== | ||
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+ | * FACS measurements for the 6 24-well plates from two days earlier were done to measure YFP expression for the 48 different clones (AAVs!)in different kinds of cells. Cells were trypsinized (100 microliters trypsin) and then 500 microliters of buffer were added. Each well was measured for 1 min. The results showed no YFP expression for 47 of the samples in HepG2 and Huh-7, whereas one sample was positive. This was confirmed further by microscopy. For the primary hepatocyes no sample showed any positive result. | ||
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+ | ==17/10/2010== | ||
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{{:Team:Heidelberg/Single_Bottom}} | {{:Team:Heidelberg/Single_Bottom}} |
Revision as of 10:58, 27 October 2010
October01/10/2010
02/10/2010
03/10/2010
05/10/2010
- Washing the flasks with the medium they contain, and collecting the media from 13 flasks in 500 ml corning conical centrifuge tubes. - The cells were pelleted by centrifuging at 1500 rpm for 15 min at 4 ͦC - Discard supernatant - Washing: Resuspend the cells in 1X PBS and transfer to a 50 ml falcon - Pellet the cells again by centrifuging at 1500 rpm for 15 min at 4 ͦC - Discard supernatant
- Add 20 ml lysis buffer to the cell pellet - Put in liquid nitrogen for 5 min - Thaw at 37ͦC The cycles were repeated for 5 times, and the sample was frozen for next day. 06/10/2010
- The sample from previous day was sonicated in a sonication bath for 1 min 20 sec. - 50 units/ml benzonase were added to destroy RNA and DNA from non-AAV sources, which could later interfere with qPCR. - The sample was incubated at 37 ͦC for 30 min, and vortexed every 10 min. - The sample was then centrifuged for 15 min at 3270 xg - The supernatant was transferred into a new 50 ml falcon
- A Pasteur pipette was plugged into a Beckman quick-seal centrifuge tube - Using a 1000 µl pipette, 20 ml of the virus suspension were added the gradient was poured through the Pasteur pipette in the following order: - 7 ml 15% Iodixanol solution - 5 ml 25% Iodixanol solution - 4 ml 40% Iodixanol solution - 4 ml 60% Iodixanol solution - The Pasteur pipette was carefully removed and the tube was sealed. A tare tube was prepared in the same way. - Ultracentrifugation of the sample was carried out at 50,000 rpm for 2:30 hours at 4ͦC. - The virus-containing phase is the 40% iodixanol phase, which was sucked out using a syringe and a needle. The AAV sample was divided into aliquotes, one of them stored at 4 ͦC for qPCR the next day, and the rest were frozen in liquid nitrogen then transferred to -20 ͦC. 07/10/2010
- AAV MOI 100, 10 and 1 were used on Huh-7 cells plated on 6-well plates, and the viruses were allowed to infect cells for 1 hour and 30 min. - Adeno-virus 5 (MOI 10) was added then in a Biohazard II lab under the superviosion of lab personnel. - Cells were incubated in the Biohazard II lab. 08/10/2010- A non-ITR AAV helper vector was obtained and maxi-preped to clone into it the cap genes from the 50 single clones picked.
09/10/2010- The non-ITR vector was cut with AscI and PacI, and so was the pTR-UF3 containg cap genes from the 50 clones. The bands were purified from the gel.
10/10/2010
11/10/2010
13/10/2010
14/10/2010
- two 24-well plates HepG2 cells - Two 24-well plates Huh-7 cells - Two 24-well plates primary hepatocytes
15/10/2010
16/10/2010
17/10/2010
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