T/A cloning again. To Kinase or Not

Dr. Duncan Clark duncan at genesys.demon.co.uk
Wed Jan 17 09:22:05 EST 1996


In article <821806773.7711 at pplros.demon.co.uk>, Michael Dalrymple
<dalrymple at pplros.demon.co.uk> writes
>In article <4dg4u5$3cc at bisance.citi2.fr> Francois-Xavier Maquart,
>maquart at bisance.citi2.fr writes:
>> Should I kinase PCR products
>>(or oligos) for T/A cloning? Either Promega pGEM-T vector system kit
>>or Invitrogen TA cloning system kit do not discuss this problem.
>
>Put simply, yes.

No. If you phosphatase a vector you can still ligate in DNA fragments
providing they have their own 5'phosphate. In this case the unkinased
PCR product is 5'phosphate minus and the TA vector is 5'phosphate plus
so ligation procedes fine. You only need one 5'phosphate to get
sucessfull ligatation.  

Duncan
     
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