Hello,
I am in the step of optimization. Initially, i reduced the number of cycle in order to get low amount of PCR product per loading well, but that did not show any bands (left image in the attachment). Then after increasing the number of cycle there is a thick smear following the bands (right image). Even after loading small amount of PCR product i am getting the same pattern. The DNA conc is normalised (5-8ng/ul). Also i have tried increasing the annealing temperature to reduce the secondary amplifications. Kindly suggest.
Regards,
Neha
Hi..
I think you can lower down the concentration of Mg ions, also too much template also leads to smearing but you have mentioned low DNA concentration (try to optimise that). You can still try to increase the annealing temperature with less PCR cycles
Hope it works!
Dear Roy
Try to use less Mg2+, less enzyme and a lower concentration of DNA as well as less time for annealing temperature. Let me say that first of all, check the quality of DNA.
Best regards
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