Directional cloning problem - (Mar/10/2006 )

Hi,

I need your opinion on this.

I have a mammalian expression plasmid in my lab. This particular vector was a gift from my supervisor's friend in the UK. According to accompanying literature, the vector is a pUC 19-based construct with an additional mammalian expression promoters from Harvey Murine LTR inserted at the EcoRI sites. The insertion of the LTR rendered the pUC 19 vector with ability to express proteins in mammalian cells system.

For my project, I plan to use this pUC19/LTR as a carried of a human papillomavirus (HPV) gene fragment into HeLa cell line.

I first PCR-amplified the gene fragment. The forward primer that I used has a BamHI recognition site + 3 additional nucleotides at the 5' extension. The reverse primer has a HindIII sites + 6 additional nucleotides at the 5' extension. The number of nucleotides that I put up at the 5'extension is based on the reccomendation by NEB websites. My PCR product size is 1.1 kb.
I've sent my PCR product for sequencing, and the presence of of both BamHI and HindIII at the gene fragment are confirmed.

After PCR, I clean the PCR product using QIAGEN Gel Clean Up kit. I then double digest my PCR product using BamHI/ HindIII enzymes so that my gene fragment will have a cohesive end at both sides. Both restriction enzymes are from Fermentas.

For the pUC19/LTR plasmid, I did a miniculture then extract out the plasmid using QIAGEN's QIAprep Minispin Kit. I double digest the plasmid using BamHI/HindIII.

I try to ligate the insert into my vector using Promega's Ligafast kit, and all I got is just a self-ligating vector clones.

I did a 1:5 and 1:10 vector:insert ligation ratio but they both produced the same self ligating clones.

My cloning result really baffles me. How could this happen? The vectors are cut using 2 different RE that produced 2 different recognition sites. The only explaination that I can come up with is that one of the enzyme is not working, and I only end up with vector that have 2 complimentary cut sites. But I have religously followed the manufaturer's instruction. Fermentas has a dedicated websites telling users what kind of buffers to use for double digestion, the incubation condition, bla..bla..bla... all the usual stuff.

I ve tried to play around with the enzyme volume, and I ve tried to prolong the digestion period (up to overnight) but I still get self ligating clones.

I m at my witts end, and need help badly. Please tell me what else is there to be done.

See how close the BamHI and HindIII RE sites are within your vector. If they are within a 'multiple cloning site' than they may physically be too close for an efficient double digest. It that is the case try a sequential digest.

Also, gel purify your vector and pcr fragment after your restriction digests. This well clear all nucleotides and fragments and uncut vector for the final ligation.

Hope this helps.
ian

hi
2 adds :
first, NEB recommends sequential digestion for these enzymes... so here may be a tough point.
second, i've noticed that my ligations are now far better when using a pure agarose for gel purifying my fragments. I now try to avoid gel purifs as i can.