Hello, everyone,
I am setting up the digestion and ligation reaction of a plasmid and DNA insert to be transformed into E. coli.
Before proceeding with the transformation I wanted to verify the success of my ligation by PCR with primers appearing on the insert (FW) and the plasmid (RV). The ends created by the two enzymes with which I perform digestion (NdeI and NotI) are sticky.
I ran one reaction with plasmid+DNA+ligase and another in which I inserted DNA+plasmid WITHOUT ligase to evaluate how the failed ligation would show up in the gel.
Once I set up the PCR on both reactions I mentioned, I tried running it on gel, but unexpectedly I have a band of expected size even in the pcr reaction where I had previously not inserted the ligase. How is this possible? Could I have contaminated or is it possible that there is self-ligation activity since they are sticky ends? Thank you.
If both of your primers are situated inside the insert sequence then they will amplify in both ligated and not ligated reaction mixes. To distinguish inserted template from unincorporated insert you must design primers so that one primer is within the insert and the other is within the plasmid . Then only inserted template can amplify
Why did you set up a reaction with no ligase? The goal is to create your plasmid with your insert.
I can see it being useful if your mentor wants you to really understand all of the steps in a protocol & do every single control one time. But most folks only set up steps that get you to the desired product.
As Paul Rutland says, you need to set up one PCR reaction with one primer on the backbone & one in the insert. Or PCR using primers that "span" the insert site - those will give you a small product if the insert if the insert is missing and a larger product if the insert is present. You can determine the exact size with what you know about the insert & the primer map.
Good luck!
Yes, that is what's happening. "PCR-mediated ligation" is a thing. People do it on purpose ;-)
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