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angelo's picture

I have troubles in getting my ligation. What is the minimal amount of vector I should digest first and ligate?


Fraser Moss
Fraser Moss's picture
It isnt a question of how

It isnt a question of how little you should digest.

You should digest enough, so that you can then clean it up from the restriction enzymes, then have enough to put into the ligation reaction.

You should assemble the ligation reaction with a molar ratio of linearized plasmid : insert of 1:3. NB this is molar ratio NOT mass.
So if your plasmid is 3kb and your insert 1kb, you should ligate an equal mass of vector and isert to get the right ratio.

Make sure you have dephosphorylated your vector too so that it cannot religate on itself if you have opened it with a single cut, or in case one of your enzymes did not cut properly in a double cut.

You can easily work with as little as tens of nanograms of cDNA in a ligation to get enough to transform and grow.

Galicola's picture
It also depends on the

It also depends on the efficiency of your competent cells. The more efficient the cells, the less vector is needed.

I work with a minimum of 50ng vector for ligation, ususually 100-150ng. (for digestion i usually take at least 2 micrograms)

If you have a problem of self ligation, i suggest you do these things:
1: lengthen the digestion time, even up to over night.
2: run the digested vector on gel and see if you have only one band (linearized) and extract it from the gel to get rid of undigested vector.
3: dephosphorylate your vector (i de-P for at least 1 hour).

to increase transformation yeild, do heat inactivation to the ligase after the ligation reaction. I don't know why it works, but it does.