Question

Name all possible unwanted products of the ligation reaction you will be performing in the lab. The reaction will involve incubating blunt-ended plasmid and blunt- ended PCR product with DNA ligase and ATP. -primer dimers -self-ligation of inserts -ligation of insert with primers -ligation of primers with vector -ligation of vector with 2 inserts -self-ligation of vector

the options are with a : -

Answer 1

Before ligation, the vector is digested with an enzyme which create blunt ends, for example Sma1. The insert is parallelly subjected to phosphorylation after PCR amplification followed by PCR clean up. The aim of PCR clean up is to remove the unwanted bands and primer dimers. Specific band of the insert is taken out from the gel and eluted. Phosphorylation is done to provide a phosphate group to the 5' end of the amplified product for the ligation with the plasmid to happen.

These two products, the phophorylated amplicon and the digested plasmid is subjected to ligation. Here, we may get

1. Self ligation of the insert, because after phosphorylation the insert can form the phosphodiester bond.

2. Ligation of vector with 2 insert: If self ligated insert is ligated to the vector.

3. Self ligation of the vector. As no insert is there, the vector itself will ligate.

Primers forming dimers or primer getting ligated with insert/ vector will not happen in this scenario as the primers are removed during PCR clean up.