I followed the standard protocol of Qiagen DNeasy Blood & Tissue Kits to extract DNA of eel fins of various species and amplify DNA by PCR and yet after PCR and gel electrophoresis, the amplified DNA is still not detected. This occurs randomly in a way that: DNA of some replicates of a species can be detected but some cannot be detected; even if genomic concentration is high, amplified DNA is still not detected and when conc. is low, amplified DNA is then detected. This can also happen the other way around. As mentioned, the occurrence is completely random. Is there something wrong with the machine itself? Do I need to modify the temperatures? I used universal primers.
Dear Nyca, for me, the best way to check is PCR optimization with temperature (Ta) gradient (I prefer in two step real time protocol 56, 58, 60, 62, 64oC annealing/elongation step) and Mg concentration (1, 2, 3 mM in the case of Mg free mastermix of course). After that I usually I combine temperature gradient with oligonucleotide concentration 100 nM, 250 nM, 500 nM. May be it helps You.
it may be that you are not completely removing pcr inhibitors. When a sample does not work try using less dna ( therefore less inhibitor) and also more Mg as many inhibitors remove the Mg needed by the enzyme. Ethanol precipitation of dna refusing to amplify may also help
I will recommend the following:
1. Use NEB Tm calculator (https://tmcalculator.neb.com/#!/main) to calculate the optimal annealing temperature of your primer.
2. Try to run 1uL of the sample DNA on gel before amplification to ensure that your sample DNA is not degraded (i.e presence of clear bands)
3. as Paul Rutland have stated, if ethanol is present in your sample, it will interfere with your PCR. TRY AND CONCENTRATE THE DNA IN A SPEED VACUUM to dry out the ethanol
All the best!
Paul Rutland Alice Eseola I did check the bands beforehand before performing PCR but the amplified DNA is still not detected. I will try to increase the Mg2+ conc. and do what Andrei S. Babenka has suggested.
Anymore suggestions / solutions are more than welcome!
Could you present a gel(s0 after optimization (with DNA ladder if you can), please?
After DNA isolation gnomic DNA concentration and purity was what...? If it was good then go for gradient PCR.
Dear Nica,
Check your DNA quality by Fluorometer/Nanodrop/gel electrophoresis. If the quality is good then follow the gradient PCR according to the thermocycler. You can use commercially available master mix for better results.
I will try Andrei S. Babenka but it will take a while since I am currently not in campus... Anil Kumar Sah Akhilesh Kumar the genomic concentration is good so I will try perform gradient PCR as well
Paul Rutland Hello! In the protocol, the amount of Mg to add is 25 μl.. you reckon how much more Mg to add to overcome the inhibitors?
Go for gradient PCR first and standardised annealing temperature then go for amplification.
Since I am suggesting pcr inhibitors as a possibility add twice as much MG at first.If the pcr works it will produce non specific bands as well and then you can reduce the amount. You could also try adding half of one dna that does not work to a dna that does always work.If the pcr fails then it is likely that there is an inhibitor present ( that experiment at normal Mg concentration)
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