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Here is the problem - I have mouse samples from archive materials and want to amplify fragments of 4 different mRNAs (i.e. cDNAs), using two step RT-PCR. I designed the primers using Primer 3 Plus, and checked for secondary structures and interactions in Premiere Biosoft. The length of primers is 20b (only one is 19), Tm should be ~60 deg (+-1 deg), the length of amplicons is ~ 100 bp. I need these primers for qPCR.
Before this experiment all samples were checked for quality of RT-reaction - I amplified fragment of housekeeping gene with length 316 bp and spanning 1 intron.
Two days ago I reconstituted all 4 primer pairs in this order - 1, 2, 3 and 4. I prepared the primer work dilutions in the indicated order, the 4 reaction mixtures as well, and loaded everything in this order. Everything was done in this order.
The result: primer pair 1 and 3 were fine (i.e. amplify only fragment with appropriate length and empty negative sample).
Primer pairs 2 and 4 give the amplicon and another band with app. length 50 bp, which is present also in the empty line.
The gel shows primer pairs 3 and 4, as follows:
1. Low range DNA marker (700-25 bp);
2. Four samples with pair 3 and empty sample with pair 3 (primer 1 looks exactly like this);
3. Four samples with pair 4 and empty sample (primer 2 looks exactly like this). There is some problem with the third sample.
Well, when designed the primers I choose Tm~60 degrees, but when I received them, Tm indicated by the producer was different. However, I run my PCR with annealing temperature 55 deg, as I always do.
What do you think is this? Are these two pairs contaminated somehow? Is it possible mouse primers to amplify human DNA? So small fragment?
Yesterday I repeated the PCR with new aliquots of the problematic pairs (2 and 4)versus the previous aliquots to see if it is a technical mistake - the result is the same :-(
