Tranformation cell harvesting - (Sep/07/2009 )

After transformation, I grow colonies in 5 ml LB and then in 200 mls of LB. Then I centrifuge at 4500 rpm in Beckman Centrifuge for 20 mins. After that I decant the LB and am told that I can store the cells at -20C. The problem is I cannot store cells in the bigger centrifuge vessels as others in the lab need the centrifuge vessels. Can I wash the cells out (by pipetting up and down) using WATER into a 50 ml tube. Next day, I would centrifuge and pour the supernatant water out. Then add the lysis buffer and continue with my maxiprep. Is this a viable strategy? Does water interfere with the cells/lysis buffer etc in any way?
THANKS!

You added a hypo-osmotic solution to the bacteria, they won't like it. I would freeze them as a pellet if possible, or in 5% glycerol in medium.

Thanks. The problem I cannot just decant and freeze the pellet in the centrifuge vessel. I think I will just resuspend the cells in ~25 mls of LB and store in a 50 ml tube.

Or, you could resuspend the cells in the lysis solution, transfer to a 50 ml tube and freeze it. Save yourself one step tomorrow.

you mean I should resuspend it in P1 (qiagen) buffer, right? because I thought that we should not incubate for more than 5 minutes in lysis buffer as that might denature the DNA. Please advice.
One more qs: when I freeze the cells at -20C either in LB or in a buffer (as per your advice, please let me know PI or P2), the liquid will freeze as well. So next day, I will have to basically thaw it out, and centrifuge again to get the cells as a pellet. Is that correct? And also, does this freeze/thaw affect the cells?

arnabde2000 on Sep 8 2009, 02:46 PM said:

The P1 solution is just for cell resuspension, and to give the RNase a happy home. One of our handbooks gives the recipe as 50 mM Tris, pH 8.0, 10 mM EDTA, 100 ug/ml RNase. If you're concerned about the stabiility of the RNase on freezing, resuspend in the non-RNase components, then add some fresh P1 on the day.

The freeze-thaw will lyse cells, but as you are going to hit them with SDS and NaOH, a little bit of prior lysis isn't going to make too much difference. As for which buffer to use, definitely don't use P2 for storage!