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Tranformation cell harvesting


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#1 arnabde2000

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Posted 07 September 2009 - 11:26 AM

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!

#2 bob1

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Posted 07 September 2009 - 04:16 PM

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.

#3 arnabde2000

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Posted 07 September 2009 - 04:44 PM

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.

#4 swanny

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Posted 07 September 2009 - 06:35 PM

Or, you could resuspend the cells in the lysis solution, transfer to a 50 ml tube and freeze it. Save yourself one step tomorrow.
Heart disease kills more women than breast cancer, but heart attack symptoms differ from men's symptoms. Get to know your heart... it could save your life.

#5 arnabde2000

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Posted 07 September 2009 - 08:46 PM

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?

#6 swanny

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Posted 07 September 2009 - 09:04 PM

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?

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!
Heart disease kills more women than breast cancer, but heart attack symptoms differ from men's symptoms. Get to know your heart... it could save your life.




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