1. (1pt) You have 8 plasmid preparations you want to digest with BamHl and Xhol.
ID: 207866 • Letter: 1
Question
1. (1pt) You have 8 plasmid preparations you want to digest with BamHl and Xhol. Assume that both enzymes work well in Buffer 2 and both need BSA. You want to digest 1.0 pg of each plasmid. Add 5 times as much enzyme as theoretically necessary. Use the smallest total volume for each digest reaction based on your knowledge of Star Activity Assume you have access to a P2 micropipette (range 0.2 to 2.0 L). Use the table below to show how you will set up the restriction digest, NO FUDGE FACTOR Component Stock Final concentration Volume for 1 Volume for- one reaction I reactions (MM) ! concentration 10X 100pg/mL 1X | 10pg/mL Buffer 2 BSA Xhol BamHl DNA Water Total 10,000U/mL 5,000U/mL 500 ngjuL | 1.0 gExplanation / Answer
1 unit of restriction enzyme is added to digest 1 microgram of DNA in a 50 microliter reaction volume in 60 minutes. As 5 times volume is required, it will be 5 unit of restriction enzyme is added to digest 1 microgram of DNA.
Under non-standard reaction conditions, some restriction enzymes cleave sequences that are non-identical but similar, which is known as “star activity". Star activity is seen in all restriction endonucleases. The amount of enzyme should not exceed 10% of total reaction volume to inhibit star activity due to glycerol in the restriction enzyme solution.
The lowest volume used is 20 microliter reaction volume for double digestion to avoid evaportation during incubation.
Component
Stock solution
Final concentration
Volume for one reaction (microliter)
Volume for 9 reactions (microliter)
Buffer 2
10X
1X
2 microliter
18
BSA
100microgram/mL
10 microgram/mL
2 microliter
18
Xhol1
10000 U/mL
5 U
0.5 microliter
4.5
BamH1
5000 U/mL
5U
1 microliter
9
DNA
500 ng/mL
1.0 mg
2 microliter
18
Water
12.5 microliter
112.5
Total
20 microliter
180
BSA: Prepare a 1: 10 dilution to get 10 mg/mL. Hence, for 20 microliters, add 2 microliters per reaction.
Xhol1: Needed is 5U in 20 microliters.
Given is 10000 U/mL or 10U/ microliter
10 units are present in 1 microliter. Hence, add 0.5 microliters to one reaction volume of 20 microliter.
BamH1: Needed is 5 U per 20 microliters.
Given is 5000 U/ microliter or 5U/ microliter
5 units are present in 1 microliter. Hence, add 1 microliter to one reaction volume of 20 microliter.
DNA: Required in 1 microgram per 20 microliter reaction
500 microgram is 0.5 microgram. Hence, 2 microliter is added per reaction.
To prepare master mix, you need to prepare a little more extra reaction mix than required in order to overcome pipetting errors. Hence, an extra 20 microliters will be prepared.
Prepare a master mix for 9 reactions instead. Multiply each reagent with 9 to obtain master mix (MM) volumes.
Component
Stock solution
Final concentration
Volume for one reaction (microliter)
Volume for 9 reactions (microliter)
Buffer 2
10X
1X
2 microliter
18
BSA
100microgram/mL
10 microgram/mL
2 microliter
18
Xhol1
10000 U/mL
5 U
0.5 microliter
4.5
BamH1
5000 U/mL
5U
1 microliter
9
DNA
500 ng/mL
1.0 mg
2 microliter
18
Water
12.5 microliter
112.5
Total
20 microliter
180
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