I carried out a PCR reaction for a group of cyprinids fish to amplify the gene mitochondrial cytochrome oxidase 1 subunit (COI) using the pair of primers Chmf4 (5' TYT CWA CWA AYC AYA AAG AYA TCG G 3') and Chmr4 (5' ACY TCR GGR TGR CCR AAR AAT CA 3').
The amplifications were performed in 25 µl volume reactions using 12.5 µl of a mastermix, 0.3 µl of each primer (10 µM), 9.9 µl of sterile water and 2 µl of DNA template. The thermal profile was 1) an initial denaturation at 95°C for 5 min; 2) 35 cycles of denaturation at 94°C for 1 min, annealing at 46°C for 1 min, extension at 72°C for 1 min and 3) followed by a final extension at 72°C for 10 min.
The gel image I got is attached herewith. The negative control is outlined by the red box. I've got three clear DNA bands only for three samples.
Note that for another gene (16S rRNA; different thermal profile), I've got clear DNA bands for all of these samples.
Couple of things will help with troubleshooting.
Like having a DNA ladder in the image, also what taq you are using and also the product size you are expecting.
Hi Hiranya,
Firstly, have you designed these primers yourself or are these commercially available? If you have designed these yourself, you may need to do a gradient PCR to figure out the optimal annealing temperature and you may also need to test different MgCl2 concentrations in your mastermix, as that can have a major effect on your results as well.
If these are commercially available, check the literature to see if others have used these primers and what conditions they used for their PCR reactions, that could be a good start.
Also check to see what your final Taq concentration is in your mastermix, as that will affect you reaction too. Good luck!
Hope that helps.
Sanaya
Dear Hiranya,
it looks like you do have a PCR product, so in what regard do you want to "optimize" the PCR conditions? By the way, how bog is your expected product (therefore a DNA ladder would be essential in the gel picture).
It would help if you briefly describe the exact conditions you used, including the enzyme.
Moreover, did you use different tissue samples, which would probably explain that there were amplicons in some samples, if the gene is tissue specific expressed.
Please give us more info, so we can help.
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