5 replies to this topic

[lukceg1983]

Hello Dear Scientists

I have a question. I have to do colony pcr to search for the clones with my insert. I am wondering if I can use exactly the same primers which I used to amplify this fragment from genomic DNA. Is it better to use some primers binding specificaly to the vector backbone? Thanks for any advice and suggestions.

cheers

lukasz

[bob1]

There are a few ways to do colony PCR, each with some advantages and disadvantages.

1) PCR using cloning primers - advantages are that you already have the primers and conditions worked out.  Disadvantages are that the PCR probably isn't hugely efficient as you can have some quite odd g/c contents etc at the start/end of genes.
2) PCR using sequencing primers - advantages are that you know these primers will work and shouldn't give false positives.  Disadvantages: often they are quite far away from the cloning site, so can make products huge and may not be very efficient.  Too much DNA can be a problem for these too.
3)PCR using one sequening primer and one primer anchored in insert sequence - advantages you can make the product as big or as small as you like. Disadvantages are that it is prone to false negatives - absence of band doesn't necessarily mean there is no insert, just that the PCR may not have worked.

[lukceg1983]

Hi Bob1

Thanks a lot. As always your reply is very fast and accurate:)
cheers

lukasz

bob1, on 29 September 2010 - 04:05 PM, said:

There are a few ways to do colony PCR, each with some advantages and disadvantages.

1) PCR using cloning primers - advantages are that you already have the primers and conditions worked out.  Disadvantages are that the PCR probably isn't hugely efficient as you can have some quite odd g/c contents etc at the start/end of genes.
2) PCR using sequencing primers - advantages are that you know these primers will work and shouldn't give false positives.  Disadvantages: often they are quite far away from the cloning site, so can make products huge and may not be very efficient.  Too much DNA can be a problem for these too.
3)PCR using one sequening primer and one primer anchored in insert sequence - advantages you can make the product as big or as small as you like. Disadvantages are that it is prone to false negatives - absence of band doesn't necessarily mean there is no insert, just that the PCR may not have worked.

[philman]

When I did my colony PCR I used the same primers I used to clone the gene in the forst place, with the restriction sites on the end and all, it seemed to work fine for me for simply showing positive and negatives anyway!

[HomeBrew]

If you're going to use the insert-generating primers for colony PCR, you must first pick your presumed positive colonies off the transformation plate to a fresh plate, allow these to grow overnight, and use the fresh colonies as template in your PCR.  If you use colonies directly from the transformation plate, you'll be overwhelmed by false positives, presumably from the presence of unligated insert on the transformation plate, which will amplify.

[lukceg1983]

Thank a lot to all of you guys for your helpful advice. I realy appreciate this.

cheers

lukasz

HomeBrew, on 01 October 2010 - 03:18 AM, said:

If you're going to use the insert-generating primers for colony PCR, you must first pick your presumed positive colonies off the transformation plate to a fresh plate, allow these to grow overnight, and use the fresh colonies as template in your PCR.  If you use colonies directly from the transformation plate, you'll be overwhelmed by false positives, presumably from the presence of unligated insert on the transformation plate, which will amplify.