TA vs topoTA cloning

Gregg Silk greggsilk at aol.com
Thu Jun 18 20:05:14 EST 1998


>> Hi,
>>
>> I want to TA clone some PCR products, but these were kept at -20 C for a
>> few weeks. Would this cause problems? Are there better ways (if any) of
>> storing the PCR reactions? Also, what is people's experience with kits
>> (TA, not blunt-end, and preferably cheap)? I am thinking about getting
>> PGEM T vector from Promega, seem to be the cheapest in Europe.
>>
>> Regards,
>>
>> Denni
>>
>> Dept. Paediatric Gastroenterology
>> Bart's Hospital
>> Dominion House
>> 59 St. Bartholomews Close
>> London EC1A 7BE
>
>I have had mediocre success with PCR products frozen at -20C for even a few
>days.  I recommend using fresh products, if that's not too much of a bother.
>You could try and +re-tail+ heproducts by incubating at 72C (if Taq) with
>dATP
>for about 10 min.
>
>As for the kit, I swear by Invitrogen's Original TA cloning kit.However, it
>is
>the onlyone I have ever tried.
>
>rich

I have used the original InVitrogen TA and the new topoTA kit. Both of them
worked well, and the 5 min "ligation" of the topo kit is slick. The blue/white
screen for the topo kit is strong, although there are plenty of blues. And
although the topo kit uses a special reagent, they provide enough for 20 or so
reactions.
Gregg



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