I have run PCR and digestion (Not1-hf & Nde1) that gave good bands. The plasmid was PET-28-make123 and the PCR product was franken-flag-dsred.
Then I performed ligation and ran a gel with ligation reaction, plasmid only, and PCR product only control and no bands were present.
How do I find out what caused this?
Did you have enough DNA to visualize in the gel? Often by the time you purify your DNA, do a ligation, and then run an aliquot on a gel there might no longer be sufficient amount of DNA to see.
Barely. You can visualize a single band that has maybe 5-10ng in it, although faintly. So you should be able to see that amount of DNA if in a single band. But after ligation you might have a number of various ligation products so that each band could be faint.
I never bothered running ligation products on a gel, I just ensured that I ran the proper controls and then transformed.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line