faint PCR product

LeboMad via methods%40net.bio.net (by lmadubanya from gmail.com)
Thu Apr 22 05:47:03 EST 2010


Guys I am amplifying a specific locus on a legume genome. I get very
faint bands and these product need a further digestion in order to
reveal the polymorphism that I need to genotype my material. I get
nothing after digestion. I PCR, run the undigested product (i.e very
faint bands) and then digest. Is there anything I can do to improve my
bands intensity? The PCR is optimized at 94 for 1 min, 35 cycles of 94
1 min, 57 1min and 72 2min and finally and extention of 72 5min. My
fragments are 1.0 kb and 1.5 kb (after digestion). I use 30 ng DNA and
primers and Promega mastermix to a total of 25 ul.

can someone please suggest something.

kind regards
Lebo RSA


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