PCR off plasmid for screening - (Mar/18/2010 )
Hello, I did a colony PCR to screen for clones but nothing amplified. I know colony PCR is tricky and this doesn't mean my colonies are negative. I plasmid preped these colonies but I cannot test them by digestion because the needed enzyme is out. If I do a PCR on the purified plasmid with the primers I used for amplifying the insert originally, can I expect more conclusive results? In other words, if I don't get a product this time, can I assume that the insert is not present?
spellberg56 on Mar 18 2010, 11:13 AM said:
For the most part, pcr screening of colonies for an insert works very well and is reliable. BUT there have been occasions where the insert won't amplify during a screen. I don't know why, but have seen it enough times to not always trust negative results when my ligations have been working fine.
Yes, you can screen for the present of your insert with the same primers you cloned with. It will not tell you if your insert is correctly orientated, but you can send 3 or four plasmids to sequencing to find out.
You can screen using one primer for the vector and one for the gene which will tell you if the insert is present and if it's inserted correctly.
Here's a screening colonies thread that should help.
There are better options now for screening large numbers of colonies:
1) PCR screening
You can screen 100 inserts with prep time in a matter of hours!!!
If you have the sequencing primers for the vector and or the primers used to clone the insert, you can run this with your regular PCR reagents.
To screen for the presence of the insert use the sequencing primers for the vector.
To screen for insert and directionality use one primer for the vector and one for the gene.
I'll attach my set-up protocol that works like a champ!
Set up each well like a regular PCR reaction.
We did 10 ul rxs
4.8 ul H20
5.0 ul Master Mix
0.1 ul Primer 1 (100 uM concentrations)
0.1 ul Primer 2 (100 uM concentrations)
1.0 ul DNA from "Cells" microplate
My second choice for screening colonies for the presence of an insert is a version of the Epilyse Kit
Here there's no cell culture, minipreps, or restriction digest.
Will only tell show you size differences of empty vector and vector with insert, not directionality.
This can be done in one day. We made our own reagents. I've since left that lab, but found a paper that gives the recipes. See below.
Here's a link to a paper that gives recipies for the two solution needed:
See page 59 Section 18.104.22.168 Method 1 Ligation and Screening
If you still want to culture the colonies and prep and cut each one, I used 1ml of media in 2ml tubes, using a rich media and shaking at 37C overnight which worked fine.
A quick test to see if your pcr screening is working and to check to see if your insert is present, is to cut with any other restriction enzyme still present in your new construct to linearize your plasmid and compare sizes on a gel. I would just miniprep a few of the colonies, cut with whatever enzyme and look for bands of the right size, i.e. vector plus insert. If those are all empty, then check a bunch of colonies by pcr and go from there. If they're all empty, you've got problems upstream of the transformation.