I am trying to genotype 14 samples of plant genomic DNA for a particular allele using specific primers. The Tm of primers is 63°C. When I first carried out the PCR reaction for first 7 samples using annealing temperature as 57°C, I got the result showing amplicon bands of size 420 bp which is correct. When I carried out same procedure with same DNA template and primers, but this time along with previous 7 samples I also amplified another 7 samples. Unfortunately, I didn't find any amplicon bands in first 7 samples in which I had got positive results earlier. I am confused about reproducibility of my technique. Any suggestions?
Since you seem to be a beginner, there is a high probability of you just making a mistake in one of the procedures (forgot to put a primer, or DNA etc.). Also if you're running a gel, make sure you're doing it with the right gel density (concentration) so that your DNA migrates properly.
Thank you Yulia, but I have ran many gels in past. For genotyping, I used 1% gel and ran it for 1 hour. I will run them again, but this time instead of doing all 14 in a stretch, I will do 7 at a time.
are you saying that you got no amplification of any sample or that the previous 7 samples failed but the new samples worked please?.Was there any band visible on any sample and are you getting a small size primer dimer or is the reaction clean?
Hello paul, when I first ran the 7 samples, I got the bands in all 7. But, when I again ran those samples (same template and primers and same conditions of pcr), I could see amplicon bands only in 5 samples instead of all 7.
It is not possible to say what the problem is but two possibilities are
1.
dna has pcr inhibitors ( plant dna is famous for purifying badly with inhibitors not completely removed and difficult to quantify accurately)and small pipetting errors lead to failure
Try amplifying LESS dna with a little more Mg for the failed samples. Try half and quarter amount of dna at 2mM Mg if you are currently using 1,5mM mg
2 sample amount is too small again due to pipetting or some dna has degraded or insufficient mixing after thawing the sample after storage before pipetting. Mix the 2 samples well and amplify same,2 times and 4 times amount of dna.
how much dna (ng) are you amplifying?
I am using Bioline taq and its buffer therefore I am not adding any external Mg (it is already presentbin bioline buffer). I am using almost 100 ng of DNA.
Thank you Amar,
if your dna ia accurately measured ( many plant based pcr inhibitors also absorb in the UV) then I think that you are using too much dna so the effect of any inhibitors is enhanced. Just using less dna may make it work better. If this fails add a little more buffer if the buffer contains the Mg
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