small insert ligation

John Watson watson_j at
Thu Sep 5 08:16:24 EST 1996

Morello Jean-Pierre wrote:
> I'm trying to insert a 200 bp fragment into a 3.4 kb vector using two sticky
> site enzymes (Bam HI and HinDIII). The 200 bp insert comes from a PCR product
> which has sufficient nucleotides on each end to allow for enzyme digestion.
> So far, I've tried to either purify both pieces of DNA prior to ligation (using the
> Geneclean kit from Bio 101) or I've taken DNA straight after digestion and
> phenol/chloroform extraction. In both cases I only get recircularized vector
> although I have verified that both enzymes have digested the DNA. In addition,
> I have treated the vector with alkaline phosphatase and phenol/chloroform
> extraction prior to digestion.
> If anyone has experience with small insert (approx. 200-300 bp) ligation
> I would greatly appreciate the help.

I have done this often for fragments in the size range you describe, though not with 
those particular sticky ends.  Please check our Meth. Enzymol. chapter for detailed 

Wilkie et al. (1994). Meth. Enzymol. 237:327-344.

Good Luck.

John Watson                                                          |
Bristol-Myers Squibb Co.                                             |
watson_j at                                                     |
"If you're not part of the solution, you're part of the precipitate."|

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