I did the plasmid extraction, and ran the PCR with my primers... Then, I did the magnetic bead purification to purify the PCR product. In the attached picture you can see smearing in the lane on the right side. I read that seeing smear while using plasmid dna is common and better to use restriction enzyme to cut the plasmid. I want to get rid of it and run the qpcr.
P.S: I don't want to do gene sequencing in my case study.
Perhaps you used too much template and now it is showing in the final reaction. You may repeat the reaction with lower amounts of template; if it is a plasmid, 100 pg should be more than enough. Another alternative would be to digest the PCR with Dpn I to get rid of the template (assuming the template comes from a dam+ strain).
Hope it helps!
Perhaps you used too much template and now it is showing in the final reaction. You may repeat the reaction with lower amounts of template; if it is a plasmid, 100 pg should be more than enough. Another alternative would be to digest the PCR with Dpn I to get rid of the template (assuming the template comes from a dam+ strain).
Hope it helps!
Hi, Termeh Hesam Mahmoudinejad.
I am not quite sure of your questions and as you did not send any specific data on your reaction setup and machine programming. So, I am assuming you did a PCR with a plasmid as template and then purified your amplimer by a magnetic system. If so, I guess:
1 - Too much plasmid at the beginning;
2 - you did not cut the plasmid to break it open with a restriction enzyme;
3 - your amplimers may be arrested in conjunction with the plasmid template, appearing in your gel.
Looking into some papers and manuals, they all comment about plasmid backbone being strong enough to reanneal just after PCR denaturation step, and so, in the end of purification and visualization in gels.
My opinion is, cut it all open before PCR and do purification after cutting your product from the gel. Ok?
Hope it helps.
All the best.
Alejandro Martin Thank you so much for your reply, actually I also thought the concentration was too high and I reduced it to 100pg but there is still the same smear in the PCR product.
Thank you.
Adriano Costa de Alcantara Thank you so much for your reply, yes, this is exactly what I've asked. I guess number 1 is not the problem because when I ran the gel with the PCR product which contains 100pg the smear will be the same.
For the attached picture the PCR program was 95 °C for 3min, 95 °C for 30s, 56 °C for 30s, 72 °C for 45s, 72 °C for 5min. I decreased the times in bold font (Annealing and elongation) to 15s, but it didn't change the result.
The amplicon size is 261bp.
Thank you.
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