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Parhit Постоянный участник Москва - Сергиев Посад |
Hirt lysing buffer Hirt resuspension buffer Применяются для методики Infectious DNA Preparation <img src="graemlins/shuffle.gif" border="0" alt="[смущение]" /> |
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http://www.qbiogene.com/literature/protoco...-adenoquest.pdf |
Parhit Постоянный участник Москва - Сергиев Посад |
Если не сложно, посмотрите, пожалуста, у себя в библиотеке. Я бы приехал и отксерокопировал. Мой e-mail: parhitko@pochtamt.ru Заранее благодарен! |
Tom1 Постоянный участник |
Unfortunately our library I mean so far from you... So I try find this journal and will try send to you via e-mail Tom |
Parhit Постоянный участник Москва - Сергиев Посад |
Очень нужно. |
Tom1 Постоянный участник |
eto vidno vse pridumki firmiu Stat'ya drevnya |
Parhit Постоянный участник Москва - Сергиев Посад |
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Tom1 Постоянный участник |
Generation of recombinant adenovirus cDNAs for each of the signaling intermediates were cloned into the vector pAC-CMV (Gomez-Foix et al., 1992). Five g of the pAC were then cotransfected with 5 g pJM17 and 10 g ssDNA into 293 cells using a calcium phosphate technique. The DNA was added to 500 L of 10 mM HEPES buffer saline, pH 7.12. Thirty-two l of 2 M CaCl2 was added and a precipitate formed at room temperature for 20 - 30 min. Precipitates were added dropwise to the cells, incubated at 37°C/5% CO2 for 6 h. Cells were shocked with 15% glycerol in DMEM for 2 min at room temperature. Media was then replaced and the cell returned to 37%. Two hundred and ninety-three cells were grown in Cellgro DMEM with 10% heat inactivated fetal bovine serum, 1% penicillin/strepavitin, and 1% L-glutamine. Cells were maintained post transfection by replacement of 3 ml of media biweekly for 3 - 4 weeks until the entire plate had lysed. Lysates were collected, underwent two rounds of freeze/thawing and spun to remove cell debris. A plate of 293 cells was then reinfected with 100 l of the lysate. Thirty-six hours after addition of the potential adenoviral lysate, DNA was collected from the cells using Hirt buffer (0.6% SDS, 10 mM EDTA, 100 g/ml proteinase K). Southern analysis was performed to confirm the presence of the insert of interest. Two hundred and ninety-three cells were then infected with serial dilutions of the original lysate and overlayed with agar. Ten days later, isolated plaques of virus were collected and used to reinfect 293 cells to obtain clonal stocks of the recombinant adenovirus. Southern blot analysis was used to reconfirm the presence of the insert. Once clonal stocks are confirmed, small amounts of these lysates were used to reinfect several 293 plates of cells to obtain 30 - 40 mL of recombinant adenovirus lysate. |
Tom1 Постоянный участник |
Естно правильно надо читать не грамм а микрограмм [Текст переведён с транслита] [Текст переведён с транслита] |
watchesonline89 Постоянный участник |
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