M13/pUC sequencing primers

[Ayesha Kanwal]

Hi, I'm using Thermo Scientific insTAclone PCR cloning kit. Well, they are suggesting M13/pUC sequencing primers instead of species specific primers in colony PCR for analysis of recombinant clones. What is the purpose of these primers where as we confirm our insert by using species specific primers as well.. What if I use M13/pUC sequencing primers and not the spp. specific ones?

[relaxin]

If you are cloning a gene into expression or reporter vector, it is better to confirm the presence of insert by using vector-specific primers. Sometimes, nonspecific nuclease may chew up sequences around the cloning site, and that leads to no expression (even the insert is relatively intact).

In general, it is wise to screen colonies with vector-specific primers in the first round. Then after streaking for single colony, screen a second time with one primer based on vector and the other based on insert sequence.

Sequening primer is generally too short for PCR. You need to make a longer primer (20-21mer) using more bases around the site.

[mchlbrmn]

In a kit they can provide vector primers that have the advantage of working on any insert. However, many inserts will be too large, and/or with difficult sequence, to PCR easily, especially in an a PCR of lower efficiency because of contaminating bacterial cell debris. So, for a large insert it might be better to screen with a smaller PCR product from at least one insert primer, and then confirm positives for the full insert.

[relaxin]

Unless the insert is longer than 2.4 kb (this is the longest insert I have PCRed with regular Taq), I think first colony screen should be using vector-specific primers. This will eliminate shorter (deletion mutant) clones and those with double inserts.

[ashu330327]

Dear Sir
Can I used this primers for checking the inserts in pK18mobsacB vector?. i am sending the map of this plasmid

[relaxin]

Both M13/pUC forward and reverse primers are 22 bp long. It looks like you can use them for colony PCR. Check the Tm to determine the annealing temperature.