Question

7. You purify DNA from a miniprep and wis units/ml (1 unit = 1 hg of DNA digested per hour u to ensure complete digestion), 5X reaction butter, (b-e) to calculate how to prepare a restriction enzym a. Using the A260, what is the DNA's concentration? na miniprep and wish to digest it with EcoRI. You have DNA with an A260 of 2.0, EcoRI at digested per hour under optimal conditions, but 10 units are usually used per reaction " A reaction buffer, and 10X BSA. Calculate the concentration (a) and fill in the table late how to prepare a restriction enzyme digestion of 1 ug of DNA in 50 jul. g/ml | Reagent Volume (ul) D. Using (a), how many ul of DNA would equal 1 ug? Enter (b-e) in the tabr a C. How many ul of 5X reaction buffer should be added to the 50 pl reaction? h (b) 1 ug DNA ? Enter (b-e) in the table. (c) 5X Buffer d. How many pl of 10X BSA should be added to the 50 ul reaction? (d) 10X BSA (e) 10 units EcoRI e. 10 units of EcoRI (10,000 units/ml) must be used. How many pil is this? Water Total: 50 ul

Answer 1

a) For a ds DNA, when A260=1 and path length is 1cm, the concentraion= 50ug/ml

Thus for A260, concentration of dsDNA= 100ug/ml

b) 100ug = 1ml, therefor 1ug = 1000ul/100= 10ul

Thus 10ul of DNA would equal to 1ug

c) 5x buffer added in 50ul reaction= 1*50/5= 10ul

d) 10x bsa added in 50ul reaction= 1*50/10= 5ul

e) 10units (10,000units/ml) of EcoRI= 1000ul* 10/10,000 = 1ul

Reagents	Vol
DNA	10ul
Buffer	10ul
BSA	5ul
EcoRI	1ul
MQ	24ul
Total	50ul