mutiple DNA bands on my agarose gel - (Sep/09/2006 )
have optimised a p53 restriction pcr with hinf1 restriction enzyme. thus far we've used DNA isolated from whole blood. I either use Gentra Kit (if blood is fresh) if blood is not i use the cold spring harbor. Found better results this way. i have optimised the conditions and the pcrs have been working well
But when i isolated DNA from Cells lines(cancerous cell line) and ran this pcr, found multiple bands coming up. Now i understand that dna is dna regardless from its origion. And once a particular pcr is working , it should be optimised for any dna that should follow, COrrect? so whats happening now.
My thoughts were either
the agarose gel isnt right , cos we ran it on a 2% gel/. But i dont thnk it would answer why multiple bands
my other idea, is that maybe we putting too much templeate, that theres unspecific binding occuring cos theres too much templates to adhere to, so maybe i should drop the less dna??
could you give me some possiblities.Any help would be gratefully accepted.
Are u using specific primers for the pcr or what is it u are trying to do its not very clear....
let me say u have standardised the pcr is it???
and i usually run the pcr product on 7-8% agarose gel.... it works fine...
get me more idea on what u r trying to achievce.
Here are the criteria:
Specificity of the primers
PCR enzymes (you can use hot-start enzymes and high specificity enzymes)
Have you consider that your result might be real? Assuming everything was done correctly and you when through the whole reoptimisation procedure again, and the bands refused to go away. Then the band might be real products.
These bands might then indicate some sort of gross polymorphism and perhaps duplication. It sounds far fetched but your cells are cancerous. Those thing gather some very strange karyotypes due to their instabiliy.
My idea could be tested by PCRing a similar but unreleated cancer line. You could even do a 'negative control' and take a very different cancer line and see if your standard PCR reaction conditions produce nice single bands.
Could you post a pciture of your gel. It might help acertain if the bands are real product or messy side products.