Question

Calculate the volume of plasmid and insert needed for a ligation reaction using the specifications for NEBL T4 DNA ligase (0.020 pmol vector, 0.060 pmol insert). This reaction is ligating a 3kb vector (25ng/μL) and a 2kb (10ng/μL) insert together, and the fragments have complementary sticky ends. Round final answers to the nearest hundredths place.

Answer 1

The correct answer -

1. volume of the plasmid: 2 ml

2. insert needed = 100 mg

Step-by-step explanation:

Calculating the inserted by the formula:

`(insert\ size)/(vector\ size)*(moles\ of\ insert)/(moles\ of\ vector)=(insert)/(vector)`

for a mormal vector size using 50 mg of vector DNA per ligation reaction.

take x as required insert:

`(2)/(3)*(0.060)/(0.020)=(x)/(50)`

`(2)/(3)*(3)/(1)=(x)/(50)`

`(6)/(3)=(x)/(50)`

3x = 300

`x=(300)/(3)`

x = 100 mg

Volume of plasmid vector is :

`\text{volume of plasmid}=\frac{\text{amount of vector needed}}{\text{conc. of vector}}=(50)/(25)`

volume of plamid vector = 2 ml.