multiple products after MSP - (Sep/16/2005 )
Hello all!
I am a postgraduate student and I am studying the methylation status of a mismatch repair gene.
Although in the begining I had really good products now apart from the expected product I have non-specific ones. Why is this happening ?
Can anyone help me?
thanks in advance,
Eliza
sounds like contamination. check other posts in the molecular biology forum with regards to PCR contamination.
N
N
Thank you methylnick for your prompt response but the problem is that I always get this extra band only with methylated specific primers and only in my bisulphite treated samples never with the untreated.
Do you know why is this happening?
are there homologues or pseudogenes for the mismatch gene you are looking at? you could well be amplifying another gene as bisulfite treatment will lower the complexity of you template DNA.
However this doesn't explain why in the beginning you were getting good results. I would suggest you start with fresh reagents to rule out possible contamination.
Nick
However this doesn't explain why in the beginning you were getting good results. I would suggest you start with fresh reagents to rule out possible contamination.
Nick
Thaks I will try it!
are there homologues or pseudogenes for the mismatch gene you are looking at? you could well be amplifying another gene as bisulfite treatment will lower the complexity of you template DNA.
However this doesn't explain why in the beginning you were getting good results. I would suggest you start with fresh reagents to rule out possible contamination.
Nick
Thaks I will try it!
I know that there is a homologue in other organisms but I dont thing that this help. how can I find out if there are pseudogenes for my mismatch gene (MLH1)?
sorry i meant ortholgues of the gene within the genome.
to find out if MLH1 has pseudogenes, it should be in the genbank entry or I would just BLAT the MLH1 sequence against the human genome and see how many significant hits you get in return!
Good luck
Nick