Can you PCR a ligation reaction?

Confirmation of ligation can be done using PCR. If you did cloning in a TA cloning based vector then use universal flanking primers (Vector based) to your insert. As a reaction you can take 1-2 ul of ligation product and can perform PCR.

What is ligation PCR?

Ligation-mediated polymerase chain reaction (LM-PCR) is a genomic analysis technique for determination of (1) primary DNA nucleotide sequences (2) cytosine methylation patterns (3) DNA lesion formation and repair, and (4) in vivo protein–DNA footprints1,2,3,4.

What is a ligation reaction?

In molecular biology, ligation refers to the joining of two DNA fragments through the formation of a phosphodiester bond. An enzyme known as a ligase catalyzes the ligation reaction. In the cell, ligases repair single and double strand breaks that occur during DNA replication.

Can you ligate PCR products?

Yes, I do this all the time. Put your restriction sites into the PCR primers, with 5 or 6 junk bases between the 5′ end and the restriction site. I usually do a PCR cleanup, then digest, then gel purify. You should also gel purify your digested vector.

How do you know if a ligation worked?

You may run the ligation product on the gel to see if it worked. You should see multiple bands of higher molecular weight than your empty vector, as well as a band of the same size of the insert, since you should be using an excess of insert. Also, before ligating you should smell the ligase buffer.

How do you test for ligase activity?

You may check the efficiency of your ligation reaction by mixing the reaction with loading dye containing SDS (final SDS concentration 0,2%) and running it on a standard agarose gel. After ligation several high molecular weight bands should appear.

How do you know if ligation worked?

What are the steps of a ligation reaction?

The three steps to form a new phosphodiester bond during ligation are: enzyme adenylylation, adenylyl transfer to DNA, and nick sealing. Mg(2+) is a cofactor for catalysis, therefore at high concentration of Mg(2+) the ligation efficiency is high.

Can you Ligate two PCR products together?

You could also digest the two PCR products with only XbaI and ligate them. Then take a small aliquot and do PCR again with the primers corresponding to the “new” ends. Purify the product and digest with the other enzymes then ligate it into the vector.

Can you Ligate together 2 PCR products with a DNA ligase?

It is certainly possible, it was common practice before PCR to ligate short adapters into vectors to get a desired restriction site. Assuming that you have isolated the fragments you want (i.e. without the bits you don’t want):