I had restricted my insert and vector with EcoN1 and Xho1. After ligation, I had screened the colonies through colony PCr and found them positive. After plasmid extraction, I tried restricting the plasmid again but it shows a single band (size is equal to the insert+ vector). Do EcoN1 or Xho1 lose site after cloning? Anyone has faced the same problem with this set before? Please advice.
Mutation in the ligation site is not exclusive to those two enzymes it can happen in any cloning basically. If it is important that those sites remain intact then I suggest pick another PCR positive clone.
I presume you realize that EcoN1 restriction site has variable internal sequence, does your insert EcoN1 sequence match up perfectly with the sequence from the vector? While in theory there should be a perfect match to get ligation, you might have forced an aberrant ligation event that destroys the site.
- Badges
- Science topic