Determine how much of each component that you will need to add for each reaction

Question

Determine how much of each component that you will need to add for each reaction. Keep in mind that the final volume for each reaction is 20 microliters. Show all work.

Uncut DNA Component oncentration Stock Amount to Add to Reaction Mix Plasmid DNA Reaction Buffer Enzyme Water 50 ng/uL 10X N/A N/A Final Amount 200 ng NIA N/A Digest with Avall Component Plasmid DNA Reaction Buffer Avall Enzyme Stock Concentration 50 ng/uL 10X 10 Units/ul N/A Amount to Add to Reaction Mix Final Amount 200 ng 1X 10 Units N/A Water

Explanation / Answer

Final volume = 20 uL
C1V1 = C2V2
V1 = C2V2/C1

Uncut DNA:
Plasmid DNA:
Stock concentration = 50 ng/uL
Required amount = 200 ng
Rquired volume = 200/50 = 4 uL
Buffer:
Stock concentration = 10X
Required concentration = 1X
Total volume = 20 uL
Required volume = 2 uL

AvaII:
Plasmid DNA = 4 uL00/50 = 4 uL
Buffer = 2 uL
Enzyme = 1 uL
Water = 20 - 7 = 13 uL

EcoRI:
Plasmid DNA = 4 uL00/50 = 4 uL
Buffer = 2 uL
Enzyme = 1 uL
Water = 20 - 7 = 13 uL

AvaII and EcoRI:
Plasmid DNA = 4 uL00/50 = 4 uL
Buffer = 2 uL
AvaII = 1 uL
EcoRI = 1 uL
Water = 20 - 8 = 12 uL

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