Question

b. Generate three ligation reactions with 1:1, 2:1 and 3:1 molar ratios of the insert and the vector DNA. Your ligation should include all components in a 30 µL reaction. Keep the vector amount fixed at 60 ng per ligation reaction. You are provided with 10 x ligase buffer and DNA ligase (0.5 U/µL) to set up your ligations.

Answer 1

In princile cloning in any vector is done by cleaving of circular plasmid DNA with one or more restriction enzymes and then ligated to foreign DNA of interest with compatible terminals. This recombinant plasmid is then transformed into an appropriate strain of E.Coli. Screening of resultant transformed colonies are done by using polymerase chane reaction (PCR) method, digestion with restriction enzyme or by hybridization experiments in order to identify the desired DNA sequence.

CLONING DNA FRAGMENT WITH PROTRUDING ENDS

5' or 3' end with 1-6 basepair in length of protruding end is created by digesting the vector and the desired DNA fragment with restriction enzyme. This restriction enzyme cleave assymetrically at the recognition sequence which can be seen from the figure below:

The DNA fragment and the vector will form the linear DNA hybrid molecule when both of them have compatible protruding ends. Finally the formation of circular recombinant DNA vector formation will take place in two different steps. In first step the linear plasmid and the incoming DNA will have an inter-molecular interaction which generate an non covelent bonded linear hybrid molecule. In the second step the protruding termininal of this linear hybrid are joined together by an intra-molecular noncovelent interaction forming recombinant circular DNA molecule. Anneling between 5'-phostpate and 3'-hydroxyl residue on linear hybrid molecule which allows DNA ligase to form phosphodiester binds, this could be more clearer by the digramatic representation as shown below:

The ligation reaction for the formation of high concentration of recombinant plasmid need to be designed with care which is not that simple. Because the first intermolecular reaction stage requires high concentration of DNA nad the second intramolecular step works effectively when DNA concentration is low. However, according to Bercovich the general acceptable rule is to take equimolar amount of DNA and cloning vector with the total DNA concentration < 10 μg/mL.

Another method by which we can increase the yield of circular recombinant DNA molecule is by digesting with two different restriction enzymes with different recognition sequence. In our question we have to use this technique, but this could be more easily visualized by the figure below:

In the above question amount of vector is constant= 60 ng

And let us assume

-vector size=4 kb

-Insert size= 2 kb

Calculating how much insert is required to be added in the ligation reaction

For example we have 4 kb vector of 60 ng and we want to ligate our 2 kb insert at 1:1 ratio then using the above equation we get insert= 30 ng

for 2:1 (Insert:Vector) then insert= 60 ng

for 3:1 (Insert:Vector) then insert = 90 ng

Ligase buffer contains ATP and the necessary salts and are usually supplied as a 10X solution by the company. 1 μL ligase and ligase buffer per reaction is commonly sufficient for the reaction. And to make the final volume of the reaction to 30 μL we can add all the required volume and then bring the volume up to 30 μL with molecular grade water.