I am a really worried student who is trying to get back the amplification products which at one time were very good. I am in the process of optimizing a Nested PCR for my organism.The primers are as follows:
For the first cycle:
F primer: TCAAGCTTATTGCTAGTGCAATGTCTGC.
R primer: AGGGATCCCTGCTGCTGTGCTTGCTGCG
For the second nested cycle:
F primer: GATCAAGCTTCCTCAGCCTACTATAATGCC
R primer: CTAGGGATCCCGACAGAGCACTATTAGGC
The cycling conditions used for both the first and second cycles is
94o C – 5 mins
94o C – 30 secs for 35 cycles
57o C – 2 mins
70o C – 2 mins
70o C – 10 mins extension
The expected product size is 1003 bp in the first cycle and 483 bp in the second .I have used the reagents in the following concentration:
Water: 13.2μl, MgCl2 : 0.6 μl, Buffer :2 μl , dNTP:0.8 μl , F primer: 0.4μl , R primer0.4 μl , DNA:2 μl, Enzyme:0.6 (Taq polymerase II) μl (Total volume: 25 μl )
I used to get very good bands from clinical samples but suddenly one day everything was smears, Initially I thought it could be a problem with one of the reagents though I could’nt find which one. After replacing the reagents and the primers and repeat extraction of DNA I am getting primer dimers with the same positive sample. I have doubts as to the annealing temperature and the extension time of the enzyme.Since I have tried so many alternatives I am at a loss for ideas. Does PCR need optimization with every batch of new reagents? What could be the possible reason of this sudden reaction? Please help!!!