subcloning pcr bands

Joe Chou jchou at cgl.ucsf.edu
Tue Jul 23 11:31:33 EST 1996


egagmp at ac.upc.es (Enrique Graziano-Areste) writes:

>		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 might want to include more details about how you are trying to
clone your PCR products. Are you blunting them and trying to clone
into a blunt site in bluescript? Do you try to generate sticky ends
on the PCR product?

I've had major problems in the past cloning PCR products, but now
have the best success rates using two brute force strategies:

1) incorporate restriction sites 9 bp IN from the end of the primer.
   This results in somewhat long primers, though:
   5' - 9 nt::6 nt site::21 nt - 3', for a total of a 36 nt primer

   I gel purify the PCR product, cut it a long time with the
   restriction enzymes to generate sticky ends, sometimes gel purify
   yet again, and then clone it into bluescript.

2) Gel purify the PCR product, TA clone it using Invitrogen's TA
   cloning kit, then cut it out using the incorporated restriction
   sites.

Good luck,

Joe
   
--
- Joe Chou  (jchou at cgl.ucsf.edu)
  http://devbio-mac1.ucsf.edu/
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