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 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
GATCTTGCTCCAGCCGGATTCAGAGAAATTTAGCGGGAAAGGAGAGGCCAAAGGCTGAAC
CCAATGGTGCAAGGTTTTACGGTTCGGTCATCCTCTGTCCTGACGCCGCGGGGCCAGCGG
GAGAAGAAAGCCAGTGCGTCTCTGGGCGCAGGGGCCAGTGGGGCTCGGAGGCACAGGCAC
CCCGCGACACTCCAGGTTCCCCGACCCACGTCCCTGGCAGCCCCGATTATTTACAGCCTC
AGCAGAGCACGGGGCGGGGGCAGAGGGGCCCGCCCGGGAGGGCTGCTACTTCTTAAAACC
TCTGCGGGCTGCTTAGTCACAGCCCCCCTTGCTTGGGTGTGTCCTTCGCTCGCTCCCTCC
CTCCGTCTTAGGTCACTGTTTTCAACCTCGAATAAAAACTGCAGCCAACTTCCGAGGCAG
modDNA Sequence
GATTTCGTTTTTTTTTTAGTTTTTTTTTTAGGATATTTAATATTTTGTGAAATTTAGAGATTTTGTTTT
AGTCGGATTTAGAGAAATTTAGCGGGAAAGGAGAGGTTAAAGGTTGAATTTAATGGTGTAAGGTTT
TACGGTTCGGTTATTTTTTGTTTTGACGTCGCGGGGTTAGCGGGAGAAGAAAGTTAGTGCGTTTTT
GGGCGTAGGGGTTAGTGGGGTTCGGAGGTATAGGTATTTCGCGATATTTTAGGTTTTTCGATTTAC
GTTTTTGGTAGTTTCGATTATTTATAGTTTTAGTAGAGTACGGGGCGGGGGTAGAGGGGTTCGTTC
GGGAGGGTTGTTATTTTTTAAAATTTTTGCGGGTTGTTTAGTTATAGTTTTTTTTGTTTGGGTGTGTTT
TTCGTTCGTTTTTTTTTTTCGTTTTAGGTTATTGTTTTTAATTTCGAATAAAAATTGTAGTTAATTTTCG
AGGTAGTTTTATTG
Make longer primers. I've never been able to get PCR to work with annealing temperatures below about 48C.
I would agree with phage.
Also how many cycles are you performing?
Nick
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.
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.
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??
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 ...
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