Protocol Online logo
Top : New Forum Archives (2009-): : Molecular Cloning

Confirmation of ligation - (Feb/18/2013 )

After ligation generally we do restriction digestion to confirm if we have got the clones and if the insert is ligated to the vector. But I found on google that many people also do PCR for confirmation. My question is, if the primers spanning to only insert can also be used for this? Whatever colonies we see on plate will be having some resistance, so either it could be ligated plasmid or empty plasmid. But could it be "empty plasmid and insert both in one colony"?? . Then this will also give amplification?

-neuron-

Occasionally you do get false +ves with using the insert PCR primers, this is usually insert that has been spread on the plate after the transformation.

A good strategy is to either use sequencing primers for your plasmid or to use one primer anchored in the insert and one in the plasmid.

-bob1-

Thanks bob! How is this method quick? There is no need to do plasmid isolation? Directly dissolve the colony in MQ and set up the PCR? And if we do the plasmid isolation first then there are less chances to get false +ves? as only insert will be lost in preps?

-neuron-

why dnt you run a gel with empty vector ligated along with the insert ligated? you could able to see a shift if the insert is over one kb ..may be around 20% of the ligation volume shd be enough to visualize the band... try this...

-GNANA-

no the insert is around 300 bp and we are not able to see it on the gel. We need lot of DNA to see that band. Even in empty vector and clone we are not able to see the difference.

-neuron-

Bob has the right idea. When you use his suggested method, you would just isolate a colony and add it directly to your PCR reaction tube by swirling the pipette within the PCR tube. I usually do this to check the ligation of my product as well as run two restriction digests. Run one digest with each of your RE sites for your insert, and pick a unique RE site in your insert and one on your plasmid. Be careful when doing your PCR colony screening, I get more false positives than anything else. I usually have to abandon that approach and just return to digestions.

Good luck.

Edit: I think you mentioned that you can not see a difference between your linear vector and your vector + insert on a gel? If I were you, I would run a very low % gel so I could isolate the ligated vector + insert band. It will save you loads of time during screening of your clone.

-jerryshelly1-

Thanks jerryshelly, you mentioned low % gel, shouldn't we run a high% agarose gel to see lower bands?

-neuron-

Oops, you are right. Run a high percent gel.

-jerryshelly1-