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I did pcr for Toxoplasma genotyping with GRA6 gene. my primer sequences were forward 5-GTAGCGTGCTTGTTGGCGAC-3 reverse 5-TACAAGACATAGAGTGCCCC-3. PCR was done in a 25μl reaction mixture. I used 10pmol primer, 1.5 mM MgCl2, 0.2 mM dNTPs, 1U of taq polymerase and 2.5μl of TAq Green 5X buffer. I did not get the specific band at 800bp but a clear band near 10000bp. could any of you think of a reason? PCR conditions were one cycle of 5 min initial denaturation at 95C followed by 35 cycles of 94C for 30 s, 60C for 1 min, 72C for 2 min, and was ended by 1 cycle of final extension at 72C for 7 min (Fazaeli etal.,2000)

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  • $\begingroup$ Why do you use such a long annealing step? And if you only want to amplify around 1kb, why do you do an elongation for 2 minutes? $\endgroup$ – Chris Jun 19 '18 at 7:37
  • $\begingroup$ At least six research articles have given the same PCR profile. Anyway, I tried an annealing step with 30s and final extension with 5min, again, no good results. I am happy to know any modifications you would suggest. $\endgroup$ – Anji Jun 19 '18 at 7:48
  • $\begingroup$ Well, that something has been published and re-used by others doesn't mean that the protocol is good. It worked for them but probably has to be adapted. This is only one possibility. $\endgroup$ – Chris Jun 19 '18 at 8:58
  • $\begingroup$ Hello, and welcome to Biology.SE! You may want to take the tour to familiarise with this site and earn a badge. First of all, why are you using 2.5μl of Green 5x buffer? Don't you obtain a 0.5x final concentration instead of 1x? $\endgroup$ – LinuxBlanket Jun 19 '18 at 10:39
  • $\begingroup$ Second, 60C is pretty high for the annealing step, you could try with lower temperatures. Other things you can try are the touchdown PCR or use some additives such as BSA and DMSO. $\endgroup$ – LinuxBlanket Jun 19 '18 at 10:42

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