TA cloning vector

Mike Morris mike at medsun.unige.ch
Thu Aug 5 10:49:52 EST 1993


  We have recently worked through a lot of approaches to PCR cloning 
(we used to be able to do this, but then the weather changed, so no
more clones).
  Making T vector yourself is possible, but seems to invovle some luck -
a while ago, the consensus was: try it in big batches, so if it works
you have plenty!
  We have just tried the "second generation" Invitrgoen kit. I got the
same result as with the first generation - nothing doing.
  A Stratagene/Bluescript kit is in the offing, but is not yet available in
Europe.
  After working through a lot of parameters, we have come down to the 
following:  	PCR
		+ ATP, Klenow, and kinase
                clean up (we use Prepagene matrix)
		ligate into Bluescript/EcoRV/phosphatased, in pres.
			of 10% PEG;
		transform to HB101.

	Optional: can do a proteinase K after the Kl/kinase, apparently
		  to remove Taq Pol which hangs on desperately to its product;

        You should also titrate not only your insert concs in the ligation, but
	also the ligase concentration.

Happy cloning!


****************************
Mike Morris PhD      
Division of Medical Genetics       
Geneva                
Switzerland
****************************  



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