subcloning pcr bands

vilimf01 at vilimf01 at
Mon Jul 29 14:03:00 EST 1996

Recently, I have grown tired of T/A cloning and decided to go back to
blunt cloning of PCR products.  Since you are using bluesctipt, you
are either, making your own T/A vector, or doing blunts.  If you are
making your own T/A, I cant help you because I could never get it to
work for me.  For blunts, cut your vector along with SAP overnight
(high salt buffers preferred) then gel purify the cut SAPed vector
(decreases background).  PCR then run products over a TE spin column
and kinase with PNK.  Since these overhangs are so dang unstable, much
of the product is blunted anyway.  Gel purify the band(s) of interest
and blunt ligate 4hr-O/N then transform.  Another place I have run
into problems is with IPTG.  Some clones express things that are toxic
to the E coli, so not doing blue/white and using a lacIq like XL1-blue
instead of DH5alpha can help.  Good luck
						Regards		SVEN

In article <4t2fhe$g1b at>, egagmp at (Enrique Graziano-Areste) writes:
> Hello everybody,
> 		I am trying to clone some PCR bands from a 5' RACE.
> The main problem I have is that only one out of 70 white colonies
> use to have the insert, the other ones are white, but when I make
> PCR using the forward and reverse primer over the colony I get 
> only a band that is the polylinker (about 200 bp using pBLUESCRIPT).
> How can I increase the efficiency of my subcloning procedures?. If
> you need more details please do not hesitate in asking me. Please,
> help me. I am starting to think about suicide...
> 		Thank you in advance.
> 		Enrique

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