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primer flaw I'm missing? - (May/24/2007 )

Primers, primers, primers. My #1 issue. The primers I post below give no desired product whatsoever, yet yield a bit of primer dimer or some other unwanted clouding at <100bp. Product should be >400BP. Picture added. I know that this is a small amplicon for nested designs, but I wanted to start slowly. I did expect the clouding, as it routinely accompanies my successful amplifications. I am clueless as to why I can't get a product. Maybe I am missing some flaw in the design of the primers, any help would be highly appreciated!


The Tm that came with the primer documentation is very low, 40°C (they all have about the same Tm). Using a base stacking algorithm and taking into account my Mg concentration of 1mM, it turns to 50°C. I ran gradients in duplicate from 35°C glare.gif to 52°C, all no product

Primers for Heminested PCR

Forward outside Primer
TAGTTTTTTTTTTAGGATATTT

Forward inside Primer
TATTTTGTGAAATTTAGAGATT

Reverse Primer for both
AAATTAAAAACAATAACCTAAA
complementary to (TTTAGGTTATTGTTTTTAATTT)

Target
Primer sites marked

gDNA Sequence
AAGACCTCGCCCTCTCTCCAGCTCCTCTCCCAGGATATCCAACATCCTGTGAAACCCAGA
GATC
TTGCTCCAGCCGGATTCAGAGAAATTTAGCGGGAAAGGAGAGGCCAAAGGCTGAAC
CCAATGGTGCAAGGTTTTACGGTTCGGTCATCCTCTGTCCTGACGCCGCGGGGCCAGCGG
GAGAAGAAAGCCAGTGCGTCTCTGGGCGCAGGGGCCAGTGGGGCTCGGAGGCACAGGCAC
CCCGCGACACTCCAGGTTCCCCGACCCACGTCCCTGGCAGCCCCGATTATTTACAGCCTC
AGCAGAGCACGGGGCGGGGGCAGAGGGGCCCGCCCGGGAGGGCTGCTACTTCTTAAAACC
TCTGCGGGCTGCTTAGTCACAGCCCCCCTTGCTTGGGTGTGTCCTTCGCTCGCTCCCTCC
CTCCGTCTTAGGTCACTGTTTTCAACCTCGAATAAAAACTGCAGCCAACTTCCGAGGCAG

modDNA Sequence
GATTTCGTTTTTTTTTTAGTTTTTTTTTTAGGATATTTAATATTTTGTGAAATTTAGAGATTTTGTTTT
AGTCGGATTTAGAGAAATTTAGCGGGAAAGGAGAGGTTAAAGGTTGAATTTAATGGTGTAAGGTTT
TACGGTTCGGTTATTTTTTGTTTTGACGTCGCGGGGTTAGCGGGAGAAGAAAGTTAGTGCGTTTTT
GGGCGTAGGGGTTAGTGGGGTTCGGAGGTATAGGTATTTCGCGATATTTTAGGTTTTTCGATTTAC
GTTTTTGGTAGTTTCGATTATTTATAGTTTTAGTAGAGTACGGGGCGGGGGTAGAGGGGTTCGTTC
GGGAGGGTTGTTATTTTTTAAAATTTTTGCGGGTTGTTTAGTTATAGTTTTTTTTGTTTGGGTGTGTTT
TTCGTTCGTTTTTTTTTTTCGTTTTAGGTTATTGTTTTTAATTTCGAATAAAAATTGTAGTTAATTTTCG
AGGTAGTTTTATTG

-cyburn-

Make longer primers. I've never been able to get PCR to work with annealing temperatures below about 48C.

-phage434-

I would agree with phage.

Also how many cycles are you performing?

Nick

-methylnick-

QUOTE (methylnick @ May 24 2007, 05:14 PM)
I would agree with phage.

Also how many cycles are you performing?

Nick


35 cycles
and for the length, i wanted to take nick's advice and go with a 30mer but my boss asked me to reduce it.

-cyburn-

Your boss needs to rethink things in light of the high AT content of these bisulfite primers. 20 bp is fine for normal primers, but not for bisulfite ones. I can't offer advice on how to tell him this gently.

-phage434-

ask your boss to have a look at the predicted Tms, does he think PCR is going to work with primers of 44 degrees Tm??

-frozenlyse-

If you can't convince your boss (which would be the best), you can try to reduce the extension temperature to 65°C. But I am of the same opinion as phage, nick and frozenlyse - those primers are too short ...

-krümelmonster-

Thank you all for your insight!
My boss is actually very sensible, it wasn't a problem telling him about the primers at all. I had just mentioned the whole thing to explain my violation of the design rules described here in the forum wink.gif

-cyburn-