Hi guys,
Attached herewith is a 1% agarose gel picture of my PCR reaction (triplicates). As my total reaction volume for the triplicates were each 50 ul, 25 ul of the each reaction was loaded into one well. The profile of the triplicates was different. Only one reaction was showing the band of interest (indicated with a red box), while the other two were smeary. This would mean that the PCR reaction is inconsistent. I used 10ng/2ul of cDNA for my reaction, 0.3 uM of the primer pair, 0.3mM of dNTP, 0.5 U of KAPA HIFI DNA polymerase.
My PCR cycling conditions are as follows:
Initial denaturation: 95 celsius for 3 min
Denaturation: 98 celsius for 20 sec
Annealing: I did a 2 step cycling conditions, 52 celsius for 15 sec for 5 cycles, and 62 celsius for 15 sec for 30 cycles.
Extension: As the fragment that I want to amplify is around 2 kb, I used 2:30 min at 72 celsius
Final extension: 72 celsius for 5 min.
I am planning to add in DMSO, but would like to hear from you guys first before I proceed.
Please advise.
Thank you.
I would definitely want to run a temperature gradient to optimize the reaction as those are broad smears. If the replicates consistently provide different results, you may want to check your PCR machine as some of the wells may not heat/cool properly.
It looks like over amplification to me. I would try a titration of the cDNA of half, quarter and eighth of the amount that you are using and then if the result is still dirty a gradient of 1%,2%......8% of dmso is a good try to get a cleaner result. As Péter Gyarmati says a gradient of annealing temperatures if you have a gradient pcr machine is a very good idea
Paul Rutland Péter Gyarmati
Thank you for the suggestions. I tried reducing the number of PCR cycles to 25 instead of 35, the smear was gone and I could see the band of interest, albeit faint. I will try your suggestions and see how it turns out. 🙏
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