Question

Dr. Melody is preparing 30 µg of a new ‘unknown’ Lambda digest (with OgwXI), to be used for her class. She wants to set up a slight overdigestion using 3x the standard amount of restriction enzyme in a total volume of 300 μl, and has the following reagents:

l DNA @ 2 µg / µl

molecular water

10X reaction buffer

OgwXI restriction enzyme @ 5 U / µl

However, Dr. Melody messed up. This was her protocol: 15 μl λ DNA, 30 μl reaction buffer, 37 μl molecular water, 18 μl OgwXI

Which components are correct and which are incorrect?

Do you think this reaction will be successful anyway? Why or why not?

How should the reaction setup be corrected?

Show your calculations to justify your answer.

Answer 1

Dr. Melody restriction digestion protocol won't be sucecessful anyway because 10 X Reaction buffer concentration is incorrect and reaction volume is very low as compare to the 30 ug of λ DNA to digest. Restriction enzyme(90 U) is correct to overdigestion 30ug of λ DNA. There is a incomplete digestion of λ DNA.

Dr.Melody Protocol Standard Protocol (correct reaction set up)

10X Restriction Buffer = 30 ul ( Incorrect ) 10X Restriction Buffer = 10 ul

λ DNA(2ug/ul) = 15 ul (30ug) λ DNA(2ug/ul) = 5 ul (10ug)

Restriction Enzyme(5U/ul) = 18 ul Restriction Enzyme (5U/ul)= 6 ul

Molecular grade Water = 37 ul Molecular grade Water = 79 ul

Total Reaction volume = 100 ul (Incorrect) Total Reaction volume = 100 ul

Reaction Buffer is in 1X concentration and 1ug λ DNA digestion required 1U restriction enzyme little more is better for digestion. 1ug λ DNA digested in 10 ul total reaction volume. Dr.Melody required 300 ul reaction to digest 30 ug λ DNA.