Improving PCR specificity? - (Feb/29/2008 )
I am working on obligate parasites. I extracted my parasites from plant tissue (micromanipulator) and sucessfully extracted the DNA (sounds easy, but...). So here I am, running my PCR-gel, visualizing it and that moment my boss came in and asked me if I was producing DNA-ladders
So I tryed to excise the single bands for sequencing, but I failed (only 10-50bp difference) , I tried different temperatures, a touchdown PCR protocol, different concentrations of my chemicals, diluting my DNA but I always got mixed extracts.
I know the answer is easy: I should clone my PCR product....but my problem is, that our -80°C fridge decided to become a -4°C fridge so I will have to wait for at least the next two weeks before I can start cloning...
Maybe any further tips, I will have some time for experiments during the next weeks...
Ah, I forgot: one of my primers should be specific for the group my parasite belonges to....but Im not so sure anymore
A good PCR optimization can save you a lot of time. Before trying to use a touch down PCR try these steps:
1) If you are using published primers the settings that were used in these PCRs. It is always much easier to use these settings as a start. Are you targeting a conserved region for the parasites (You are supposed to get a single band)? You can check the size of your products.
2) Try increasing the extention time. Maybe your DNA does not have the time to fully transcribe.
3) Try to play with the annealing temperature. If you have a grantient PCR it will be much easier. (Higher annealing temperature increases specificity)
4) If you still find more bands than you expect reduce MgCl2 concentration. Repeate previous step
5) At some point you will get the bands that you expect. If the band(s) is too weak try the following: increase number of cycles, use a touch down approach, use a nested PCR
PS: You may have to use a denaturation temperature of 94oC in stead of 95oC. It will allow you to run more cycles.
I have to use the 18S rDNA-region, as it is the only region where enough data for a taxonomic study are published....so these organisms are quite diverse. I think the extension time is not the problem: I expect app. 600bp so 1min should do it...
Maybe more cycles, reducing the MgCl again and the 94°C will help...