I am trying to design primers to target either TagBFP or mTagBFP2 protein regions of a plasmid. However, these regions are 693 and 711 base pairs long, respectively. I understand the guidelines for designing a primer, but am having difficulty getting a good primer. My main question is how far away from the region(s) should I have my primer be? I am aiming to use these primers for colony PCR.
Thank you!
Hi Luu! If you aim to do colony PCR you can use one universal primer either forward or reverse from your plasmid DNA and design a primer accordingly in your targeted regions. It's worth noting that the total desired length for amplification should be below 1kb for colony PCR. I get the best results when I amplify around 500-600 bp.
If you are just trying to check which colonies have your sequence inserted then design one primer in the insert and one in the vector and the amplimer size 200-500 bases. This avoids getting amplimers from the vector or insert left over from the ligation reaction
you can use primer blast instead...
https://www.ncbi.nlm.nih.gov/tools/primer-blast/
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