When I run a PCR with a cloned plasmid, I get three bands ( product of 800 bp, dimeric around 1600 bp and trimeric around 2500 bp). This was for 25 cycles.
When I run the same PCR for 35 cycles, I get only dimeric, trimeric products and a product 4 times the actual size. I used the same primers I used for directional cloning.
What should I do to avoid the multimeric PCR products and why am I getting the multimers? Thank you.
Increasing the temperature of annealing in the reaction will help you to get rid of unspecific products.
the problem is that the product has self homology and the more product that you get the more multimerization you will get. I assume that the product self anneals at a higher temperature than the highest annealing temperature of the primers so it is likely to continue to happen. One solution might be to analyse where the self annealing takes place.. Separately amplify each end of the product with overlapping primer sets. Then when you have 2 products which are monomeric mix them, heat denature and re anneal and pcr without primers for one cycle and hopefully most of your product will be monomeric double stranded product
Do a temperature gradient and also mgcl2 .. U might lose the non specific bands. A lot depends on the primer designing. I use seq manipulation suite to finally check the product size before ordering the primer seq.
- Badges
- Science topic
- Similar topics
- Biological Science
- Kinesiology
- Running