A plasmid of mine has GOI in opposite orientation. I have another plasmid which has exact same RE site but in such a way that it orients my GOI in right direction. However even after multiple attempts of RE digestion, Gel extraction followed by ligation, I didn't got any colonies on my transformation plate. I also checked efficiency of my competent cell with empty vector and found no issue (got 200-300 colonies) . I would be grateful if anyone can suggest me any possible solution.
If you're getting no colonies following a gel extraction, usually the culprit is the gel extraction itself. The common issues I've found with gel extraction are:
1. If you're using UV light to illuminate the DNA, you need to work quickly with low power under a longer wavelength so the UV light doesn't damage your DNA to the point the plasmid can't be replicated in vivo. A better way is to find a blue light gel illuminator, and stain the gel with something like SYBR Safe that causes DNA to fluoresce under blue light rather than UV. Blue light does not appreciably damage DNA even after prolonged illumination, and you can also take your time cutting the slice precisely around the band. A smaller gel slice is always better for downstream extraction steps.
2. High concentrations of chaotropic salts left over from the first step that dissolves the agarose gel. Chaotropic ions dissolve agarose by disrupting the hydrogen bonding network. As agarose is so tough, this requires extremely high concentrations of chaotropes. Unfortunately, even relatively small amounts of these chaotropic ions left over in the extracted DNA can denature enzymes like DNA ligase causing them to fail. Usually chatropic salts absorb strongly at 230 nm on a UV spectrophotometer like a NanoDrop, giving very low 260/230 ratios (
I agree with the gel extraction being a possible issue. Do you use a column kit or a pellet in resin kit? If it's a column, add an extra wash. If it's a resin/slurry kit, add an extra spin and transfer.
You could also try a different ligase and make sure the buffer is fresh (it does expire, especially if it has been frozen/thawed many times or left out).
I like Quick Ligase from New England Biolabs, you can do plasmid prep, digestion, gel, cleanup, ligate and transform all in one reasonable day.
Good luck!
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