Here's my issue--I've been troubleshooting cloning for the last 2-3 months but can't seem to get any colonies. I have been able to digest the plasmid (E. coli PLJR965 ~8600bp) at a reasonable concentration using BSMBI-v2. I want to ligate my vector with a ~25bp insert using T4 DNA ligase I believe the issue lies with the ligation reaction since my digested plasmid looks good on a gel. Here's what I've tried and hasn't worked to get my vector and inserts to ligate:
- PCR purification of digested plasmid before ligation
- Heat killing restriction enzymes for digested plasmid before ligation
- varying ratio of insert:vector (3:1, 10:1, 20:1)
- using new T4 DNA ligase buffer
- 30 min ligation at RT
- overnight ligation at RT
I've run controls with transforming just my undigested plasmid and that worked, so I know the problem likely isn't the transformation. I'm at a loss for what I can try next. Any suggestions appreciated!
Try to gel extract rather than PCR purify. Check the buffer of T4 ligase (thaw it on ice) if possible add 0.25microlit of dATP. and try ligation. after ligation don't store it directly attempt transformation.
By "no colonies" do you mean literally no colonies grow after transformation or do you mean you get some colonies but they do not contain your desired insert?
If you have literally no colonies, I would try the following control:
Digest PLJR965 with BsmBI-V2 the way you would normally, heat inactivate it at 80°C for 20 minutes (do NOT purify the DNA as you will lose the small BsmBI fragment) then set up a ligation reaction as you normally would (except without your insert, since that's already present) and transform it.
The PLJR965 vector should just religate back to its original form with the original insert, and you will get a lot of colonies that contain the original vector. That would eliminate a lot of variables.
If you get no colonies or only a handful, something is wrong with either the ligation, your transformation procedure or your E. coli competent cells. Transformation/competency issues are still possible even if you get colonies with undigested plasmid, as supercoiled plasmid transformation is wildly more efficient than a plasmid that's been linearized and re-ligated.
Raj Kishore Sahoo Can I know why you suggest gel extract rather than PCR purify and how can dATP adding effect on ligation process? Thanks
May be some of your plasmid remains undigested and T4 ligase buffer comes with dATP, increasing the conc. by adding extra may have chances to ligate. You can have a try.
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