TA cloning

Hiranya Roychowdhury hroychow at NMSU.EDU
Thu Jun 18 11:07:46 EST 1998


At 11:10 AM 6/17/98 -0700, Jun Zhang wrote:
>Hi, Denni,
>
>The kit recommands fresh PCR products. They even don't recommand gel
>purified PCR product. I think the A tail is not quite stable. At least
>lower the ligation efficiency. Good luck.
>
>Mark
>
>> > 
>> 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 been able to successfully clone, into pGEMT, PCR products
that were stored for about 6 months following "gel purification".
Purification of the amplicons(s) may be the key here. I do it as a matter of
routine with all successful amplifications, so i can't comment on the
clonability of products from unpurified rxns.
        The pGEMT kits seem to work for us fine. Some batches in the past
have been less than satisfactory. Those that work/worked, continue to do so
for up to almost a year without significant problems. I aliquot the vector
into 0.5mL tubes at a 25ng/uL concentration (using 0.5xTE), such that each
aliquote does not go thru more than 3 freeze-thaws.
        In a recent brochure from Promega, there is a report on how the kit
performed under different conditions of storage and use. 

PS: Standard disclaimer applies. 


Dr. Hiranya Sankar Roychowdhury
Plant Genetic Engineering Lab.
New Mexico State University
Las Cruces, NM 88003
Ph. (505) 646-5785
hroychow at nmsu.edu




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