'GCA AAG AGA CCT CAT CTG TGG A' - WT Forward Primer
'CAG GGT TAG TAG GGG CTG AC' - Common Primer
'CAA AAG CGC TCT GAA GTT CCT' - Mt Forward Primer
PCR Protocol is attached. In brief, used as low as a 50 degree annealing temp, despite the recommended/calculated annealing temp of ~55 degrees. The WT band always shows up, but the Mt band does not.
Hi chase,
testing your primers on UCSC with its in silico PCR tool, I do not see any amplification. please test at this site (http://genome.ucsc.edu/cgi-bin/hgPcr?hgsid=717472949_9CrEfUghERKF2eLauiJ9lMm8mV2N). are you sure of your primers, did you design them by yourself or picked up in a paper?
fred
The primer designs were supplied by Jackson Labs (supplies the mice and the recommended PCR primers).'
As far as I can tell, that website works for the wildtype primer but not for the mutant band I need to find.
those primers were designed for SNP or CNP? as it is done in ARMS protocols, maybe you should introduce a control PCR (duplex PCR with an amplicon length different from the targeted ones and conditions as for those ones and WT and Mt in separated PCR) to be sure nothing wrong during PCR.
fred
Hi Chase
I am agree with Fred... U should run a duplex PCR as control to avoid any ambiguity. Furthermore, it would be better if u had your own primers designed.
Hi Mehran Akhtar and Chase Hellmer
I agree, in science it's better to test things that to trust what someone sold you, even if it's been published in higher journals.
fred
Exactly...... This has happened to me once. I used already designed primers (from published paper) which didnt give results for tetra-primer ARMS PCR. Then i designed my own primers for the same SNP and got results successfully.
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