how to improve PCR yield? - (Jun/27/2008 )

hello, everyone, I'm amplifying a fragment about 500 bp but the bands are very weak in the gel after PCR.
For some samples, there's even no visable band. however, another fragment about 200 bp works very well, the bands are very bright.
these samples are genomic DNA extracted directly from lice collected from different countires.

I tried different PCR condition, such as reducing the annealling temperature 2-4 degree, increasing the PCR cycle number from 35 to 45, using more DNA templete, but still low yield of PCR amplicons.

would you please give me some suggestions of improving the PCR yield?

Thanks in advance

Hallo, how pure and concentrated is your template, it should not to by bellow 1,9. You can use proteinase K to remove histone protein and others and ethanol precipitate your DNA
Other way is to use different polymerase. Template can by GC rich what make problems, or just komplex
good luck

hi, thanks for opening another door for me
I will try to remove histone protein and reprecipitate genomic DNA in the samples.
actually, I have designed another set of primers outside the previous ones. Hope this also helpful but I'm still waiting for the new primers

any other suggestions?

thanks

well purification is the most important thing
another is your Ploymerase are they old or new?

you could also change your Polymerase which I did once and get a good answer for 750bp fragment

Cheers,
akhshik.

Hey!!!
U can also try '' Hot start''. It helps denaturating
the DNA template. I worked for me many times