Issues with pcDNA3.1Directional cloning - (Jun/05/2007 )
Just wondering if anyone could suggest why I might be getting problems with ligation into a pcDNA3.1 Directional Vector.
I'm cloning in a 1.4kb insert into the vector, amplified up with Pfu to generate blunt ends, gel purified my product and ligated into the pcDNA3.1 vector.
I get a fair few colonies on an Amp plate, with all the controls working. However, when I screen colonies with vector primers, I get 2 bands that come up. The bands add up to the size of my product.
Our Mol Biol consultant/lab member is suggesting the vector may be digesting my product, but he doesn't have any experience with these vectors.
Does anyone have any suggestions as to what I could maybe try to resolve this.
I'd be ridiculously appreciative.
Thanks so much
Try a different set of primers. Maybe your primer sequences are similar to some regions within your ligated fragement.
Alternatively, grow the bacteria, isolate the plasmid and verify with restriction enzymes.
Hope that helps.
You should screen with a set of primers where one primer binds to the vector and the second primers binds to the insert.
A PCR colony screen should be aiming for a small product as your template DNA isn't very clean. (200 - 900bp). A colony PCR where the positive signal is a 1.4kb product is not the best of things. Taq polymerase in a colony PCR doesn't work very well past 1kb. 1.4kb is very much at the limits of its amplication ability in such a situation.
I would suggest either
1 - finding or buy an insert specific primer that can work with your vector primer to produces a small product (under 900bp). Use said primer for your colony PCR screen
2- alternative screen your colonies by minipreps and restriction enzymes.
"Vector may be digesting my product,"
Is impossible. Problems however do arise if you use expression strain E coli for your cloning. Your insert can become scrambled. Always use cloning strains for cloning and expression strains for expression. While it is tempting to build the plasmid directly in an expression strain, the headaches that sometimes arise is not worth the trouble.