I am trying to ligate my insert (800bp) into a 3.7kb vector. Both were cut with PshaI restriction enzyme, and run on an agarose gel. QiaexII gel extraction was used to extract the two, and ligation was conducted with T4 DNA Ligase + 5x Rapid ligation buffer according to the Rapid Ligation Kit from Fermentas. The ratio of insert to vector was 9:2 approximately. After overnight ligation, electroporation was conducted with TOP10 E. coli cells. A control ligation of just the vector (no insert) was also conducted. Both plates showed no growth, meaning the ligation (or transformation) was faulty. Are there any ways or suggestions on how to make this work?

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Get your control transformation working before worrying about anything else. Transform with an uncut control plasmid and optimize conditions, and do the control along with your ligation transformations.

Only when the uncut vector transformations are working, get the ligations for no-insert cut vector working.

Finally, worry about your insertion + vector ligation.

Normally I don't suggest this, but you may want to dephosphorylate your cut vector and check for the failure to ligate as a control for your insert + vector ligation. If this works, it will reduce your background a lot. Use minimal amounts of SAP or Antarctic Phosphatase.

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What do you mean, you don't normally suggest this? This should be ALWAYS done if you are not interested in screening more than 10 colonies.

@Avinash,

You don't mention if you have dephosphorylated the vector. If not, please do it as that will reduce your chances of religation. As you don't see any colonies on any plate, make sure that your transformation protocol is working. I have never used electroporation so I don't know. Give a try using heat-shock if thats possible.

You can check your transformation protocol by trying to transform an uncut vector. If that works, then your transformation protocol is working.

Now concentrate on your ligation. Honestly, I don't see any mistake in your ligations so give a try again and it may work out. You really never know what these bugs do in the tube. If you want, keep couple of µl of ligation mix and run it on the gel to check if your ligation is working at all.

Be warned with fermentas Rapid ligation kit. I have used it 2-3 times and all I ended up with is colonies without insert.. So I have stopped using it now and use the normal T4 ligation buffer (10X) which is devoid of PEG1000.

Hope this helps

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