Problem with directional cloning - Problem with directional cloning (Sep/11/2005 )

I hope someone can help.

I am trying to clone a PCR product into a retroviral expression vector. I have cloned into this vector many times before and have never encountered problems. I designed my primers with BamH1 and XHO1 sites on the ends and got reasonable amplification. I gel purified the product (using a QIAEX II kit) and cut it for 2 hours in a double digest based on NEB double digest recommendations. I have 7 bases on the end of each product before the cut site for the enzyme to latch onto. I also cut the vector and then did a CIP treatment (although it should not be necessary). I then gel purified the products and set up ligations at 1:1, 1:3 and 1:5 ratios using T4 ligase.

The vector alone plate had 4 colonies. Most of the other plates had about 10 colonies. One plate had about 30 colonies on it. I picked 14 colonies, did minipreps and cut with BamH1 and XHO1 to pop out the insert - No insert was found in any of the colonys.

I should mention that a junior tech was doing this work and got the same result. After 3 tries, I stepped into help thinking she was making some basic mistake. I used fresh ligase, fresh restriction enzymes. The only thing that was not fresh was the 10mM Tris used to resuspend the DNA from the Minipreps.

We have been fiddling with this for over a month. Any ideas would be appreciated!

Kirsten

Hi,

I ve been encountering exactely the same problem.. and i m sorry to say, havent found a solution yet. I am cloning a receptor gene into pcdna3 using an HA tagged hindIII site and a xhoI site.

I was using a template of the gene in pcr2.1, but as this wasnt working, I went back to genomic dna. This hasnt been working either, and i ve tried to test all the possibilities.

Anyway, the point is i still havent found out, but it is reassuring to think it happens to other people too!
Will keep you posted if i get some ideas

Have you done test digests to verify that the restriction sites are present in the vector? Some of the plasmid maps can have screw ups. This will also test that your enzymes are working properly. You should also verify that there are no internal sites in your pcr fragments that would prevent proper ligation.

this is a stretch, but it is possible...are you cloning a gene into the vector that is highly toxic to E coli? (I am assuming you are using E coli as the host strain). stranger things have happened and you would not get anything if this were the case


if you have ruled everything out, this is a possibility

another thing to try is a different E coli line, they are not all the same

Some chaotropic reagents (on which purification kits are usually based), such as sodium iodide, may inhibit ligation and give inferior subcloning yields.
So my suggestion is trying other purification methods for your insert, e.g. the old-fashioned phenol-chloroform extractions, especially when large inserts are involved.
Good luck.

I forgot to add: make sure your phenol is clean and not oxidized, that is: no PINK color!

When you do your test digestion, do you find the plasmid is linearized by bamHI or XhoI? Retroviral plasmids sometimes can have trouble growing dur to recombination between the LTRs, perhaps you maybe experiencing an issue with that?

a year ago, i tried to clone a fragment into pMSCV vector and i couldn't get it, although I did successfully clone a lot of genes into pMSCV vector before that. for retroviral vectors, sometimes strange things can happen because they have LTR. I changed my competent cells to invitrogen's stbl4 cell line, and all of sudden, everything begin to work again.

Indeed consider using Stbl cells from invitrogen.

Also: how much bases are there on your primers next your restriction enzyme sites? You RE will need more extra bp than the recommened 6.

Alternatively, clone your PCR product in a topo-vector, cut it out, gel purify, then ligate...