problem with PCR

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kanu
kanu's picture
problem with PCR

hello every one,

please help me out. I am not getting any results of my PCR , tried 5 months........... CHANGED primers , everything working. I think in my protocol they have used Tris- Hcl (9.0) , ammonium sulfate and tween 20, and i am using 10x commercial buffer.........can that be a reason???

angfrsan
angfrsan's picture
Hmmm. Ok if you've changed

Hmmm. Ok if you've changed the primers a few times here are the things I would try in the following order.

1) Changing the template
               this is a biggie - especially if you are trying to clone from genomic DNA - you dont say what you'r PCR is for but if its for cloning a gene or fragment - ditch the genomic template in favor of cDNA unless its necessary. If you're screening transgenics for example there might be a problem in how your isolating the DNA so look there. 

2) Lower your annealing temperature - as low as 45C if need be.
             The lower you go the less specificity you will get but you should see something.

3) Change out all your reagents.
                    Commercial reagents should work fine. If something has gone funky with them just get rid of it and start over. I would make sure whatever polymerase you are using is actually working or get a new bottle.

4) Reconsider the size.
                You dont mention the size of the PCR product you are trying to get. If its for screening and you are looking for 150-500bps this should be a snap. once you start getting 1kb and over and it starts to get dicey. 3 and 4kb start to get impossible even if you are using the Long template kits. In those cases its better to break up the project into two more manageable fragments.

Good Luck!

Angela

Ivan Delgado
Ivan Delgado's picture
Some extra advice: if you

Some extra advice: if you have access to a gradient PCR machine, use it. There is a lot you can learn from your assay if you run replicate PCR reactions a range of annealing temperatures (from around 50 to 68).

Also, as angfrsan pointed out, your template is very important. If it is not of high quality, too dilute, etc, even a very good assay will fail. If you can use a plasmid containing your amplicon as DNA template to minimize any issues linked with your template. 

To be honest I doubt that using a commercial PCR buffer would be so different from a home made buffer that your PCR would simply stop working. Actually, I would guess that a commercial buffer would work better, not worse, than a home made buffer.