PCR cloning

Guy Hermans ghermans at luc.ac.be
Wed Jul 23 04:27:21 EST 1997

> Hello.  I am trying to clone a 6kb PCR fragment into a 5 KB
> plasmid and have had no success.  I am not sure what I am
> doing wrong.  Here is a description of what I did, If anyone
> sees any
> major flaw PLEASE let me know:   I have cloned restriction
> sites (Kpn I and Xho I) into my PCR product and verified them
> by
> sequencing.  I have put four extra bases (GCGC) on the end of
> the Kpn I site and six extra bases (GCGCGT) at the end of the
> Xho I
> site.  On my vector the Kpn I and Xho I sites are 31 bases
> apart.  I set up a double digestion with Kpn I (20 U) and Xho
> I (40 U)
> using NEB buffer system I. One hour before the digestion was
> finished I CIPped the vector (calf intestinal Phosphotase).
> I
> digested for five hours.  I ran on a 1% gel and gel purified.
> I then set up a ligation:  400 ng PCR insert, 600ng Vector, 5
> ul T4 DNA ligase buffer, 1 ul DNA ligase, brought this up
> into
> 50 ul and ran overnight at 16C.
>         My vector alone produced 5 clones and I only produced two
> clones on my vector+insert, which of course were negative.  I
> really don't know what else to do.  If anyone has any
> suggestions or has had similiar problems please fill me in.

defosforylation of a vector which is cut with two different restriction
enzymes is not necessary. Check if Kpn or Xho are 5' or 3' sticky ends. If
one or both are 3' overhanging, defosforylation would even inhibit
ligation (because of sterical hindrance). When you double digested your
vector you can try to precipitate it with isopropanol to loose the
fragment of 31bp between the two sites. (before doing so warm the digest
to 65 °C for 5 minutes). Instead of gel purifying try purifying over
sephadex G50M, so you won't loose to much vector. Check a small part on
gel. I also think that the amounts are a bit too high: I usually use 100
ng vector and 40 ng insert (depending on the size). Also try to keep the
volume as small as possible (10-20 ul) so the chances of succes are higher

Good luck!

Niels Hellings, PhD student
  Ms research Unit              Immunology research group
  Dr. L. Willems-Institute      Dept. of Physiology, LUC
  University Campus             University Campus
  B-3590 Diepenbeek             B-3590 Diepenbeek
  Belgium                       Belgium
Voice  ++32(0)11/26.92.07
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