how can I detach infected vero cells from 96 well plate without lose sample? - (Nov/22/2006 )
Hi guys,
i am scraping each well with the point of the tip before pipette up and down the medium. I am spending a certain amount of time per well but we still can see some cells attached in the plate. My primary goal is to extract the gDNA these cells and then quantify the number of copies of microorganism per cell by QPCR. Can I use any solution, or even trypsin (what concentration and for how long and at what temperature) to restore the total amount of cells or losing some cells during this approach is acceptible?
thanks a lot,
-azasp2-
QUOTE (azasp2 @ Nov 22 2006, 02:07 PM)
Hi guys,
i am scraping each well with the point of the tip before pipette up and down the medium. I am spending a certain amount of time per well but we still can see some cells attached in the plate. My primary goal is to extract the gDNA these cells and then quantify the number of copies of microorganism per cell by QPCR. Can I use any solution, or even trypsin (what concentration and for how long and at what temperature) to restore the total amount of cells or losing some cells during this approach is acceptible?
thanks a lot,
i am scraping each well with the point of the tip before pipette up and down the medium. I am spending a certain amount of time per well but we still can see some cells attached in the plate. My primary goal is to extract the gDNA these cells and then quantify the number of copies of microorganism per cell by QPCR. Can I use any solution, or even trypsin (what concentration and for how long and at what temperature) to restore the total amount of cells or losing some cells during this approach is acceptible?
thanks a lot,
Yopu can try lysis buffer with detergent, say the buffer that you are using plus 0.1-1% Triton X100, and try to go through freez-thraw cycles if you dont want to scrape too much to help lift the cells.
-genehunter-1-
QUOTE (genehunter-1 @ Nov 22 2006, 03:17 PM)
QUOTE (azasp2 @ Nov 22 2006, 02:07 PM)
Hi guys,
i am scraping each well with the point of the tip before pipette up and down the medium. I am spending a certain amount of time per well but we still can see some cells attached in the plate. My primary goal is to extract the gDNA these cells and then quantify the number of copies of microorganism per cell by QPCR. Can I use any solution, or even trypsin (what concentration and for how long and at what temperature) to restore the total amount of cells or losing some cells during this approach is acceptible?
thanks a lot,
Yopu can try lysis buffer with detergent, say the buffer that you are using plus 0.1-1% Triton X100, and try to go through freez-thraw cycles if you dont want to scrape too much to help lift the cells.
sounds reasonable,
thanks,
-azasp2-