topo ta cloning
dk at no.email.thankstospam.net
Thu Dec 30 19:04:38 EST 2004
On Thu, 30 Dec 2004 19:44:30 +0000, ChenHA <hzhen at freeuk.com> wrote:
>> Hi again.
>> Thanks for the answers.
>> I decided to test the difference between recovery and no recovery.
>> So I transfected 50 µl of invitrogen top10 cells with 1µl of ligation mix,
>> and after heatshock I plated 25µl on amp plates and the other 25µl I
>> incubated with SOC at 37 for 40 min.
>> On the first plate I got around 30 colonies and on the other plate, around
>> 300. So I guess it is worth doing after all!
>The difference might be smaller than you think. At an optimum growth
>rate of around 20 minutes doubling time, only a quarter of the 300
>colonies you see may actualy be transformants. Personally I found the
>differences to be minor, and normally unimportant unless the ligation
>you do is difficult.
As I know the difference in my case was no more than 2X, I could also
think that there might be a difference between chemical transformation
(as in above) and electroporation (that I use). If "chemical" cells are
"sicker" then perhaps they are more sensitive to the weak bactericidal
activity of Amp?
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