Hi guys, I am starting with a small project for my dissertation. And the first problem I have is that I am stuck with a cloning because of my bad PCR products yield. I am having many troubles to get a decent amount of PCR products, and I don't even know if the products are correctly amplified or not. I am trying to amplify very short PCR products: 69bp and 141bp using primers which only binds to 10bp of the template, the spare 10bp contains a restriction site (for forward and reverse) plus some random nucleotides to help restriction enzyme binding when digesting.
I am using Phusion HotStart polymerase. I started using 0,5uM of primers and i got tons of random bands. I changed primer concentration from 0,5uM to 0,2uM in order to avoid primer dimers but no PCR product is shown when I did that. I am using DMSO, and the 5x Phusion GC Buffer.
Can you give me some tips please? I really appreciate any help you can provide.
Thank you.
The 69 bp fragment is possible to clone by direct synthesis of oligos, in the case of 141 bp again try to get by synthesis in these case you could use oligos of 80 bp aprox with and overlap of 20 bp and use the oligos for PCR amplification
Don't waste your time to get that very short PCR product. If you know the sequence, just synthesize it. Less than 100 bp, all company synthesize it. Of course you have to designed two terminals properly for your product as per your intention to use. That's it. Done.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques