Question

Describe the ligation reaction. Discuss the possible products of the ligation reaction. Which ones would you expect to find in your plates? 2. Compare the ligation and control plates. Are the plates different/similar? Why? Are the transformation efficiencies similar or different? Why? What happened to e coli that did not take any the vector? Drug resistance? What is the maximum transformation efficiency that can be expected? How this compares to the trasformation efficiency of your plates? 3. For the the ligation plate: White vs Blue colonies? Compare transformation efficiencies of total transformants vs. white transformants. Religation? Was the ligation reaction successful?

Answer 1

Ligation is the joining of two DNA fragments with the help of an enzyme called DNA ligase.

In molecular biology, ligation reaction is very crucial step for creating a recombinant DNA molecule. The ligation of DNA molecule could be achieved by setting a ligation reaction. Ligation reaction contain the assay buffer, DNA fragments which needs to be joined, ligase enzyme and distilled water to set the volume.

As I mentioned, ligation is frequently used to create the recombinant circular DNA molecule during molecular cloning. In ligation, generally a plasmid digested with appropriate restriction enzyme is used as vector and the linear DNA molecules as insert

Ligation product

As the ligation reaction contain two types of DNA molecule namely vector and insert, following ligation product may formed

1) Self circularization of the vector is very frequent i.e. circular vector would be the product, that will give non recombinant transformant.

2)End to end joing of the insert: That will not go in to cell by transformation

3) Recombinant circular DNA: This will occurs when there will be the ligation of vector with the insert. This will give a recombinant transformant.

In plate, we will have the recombinant and non recombinant transformants.

Comparison of of ligation and control plates

when we are looking the efficiency of formation of recombinant during ligation, we transformed host with ligation mixture (vector + insert) and control mixture ( only vector).

On the plate on which has host transformed with ligation mixture (Ligation plate), if very large number of colony observed in comparison to the plate having the host transformed with control mixture. We can say, the ligation reaction has facilitated the ligation of vector and insert and self circularization was very low.

Transformation efficiency would be different. It depends on the how successfully ligation has occurred. If ligation has occurred with good efficiency and leads to the formation of recombinant, then the transformation efficiency of the ligation plate would be high or vice verca also true. The high transformation efficiency for ligation plate in comparison to the control plate would indicate the success of ligation reaction.  

E.coli did not take any vector, the will not be able to grow in plate containing antibiotic (used as selective agent).

Transformation efficiency is defined as the total number of transformants obtained per microgram of the total DNA used during transformation experiment

Using the above formula transformation efficiency may be calculated for the different conditions and may be compared.