You want a 50 µL reaction volume containing 1X reaction buffer, 1 µg DNA, and 10...

You want a 50 µL reaction volume containing 1X reaction buffer, 1 µg DNA, and 10 Units of enzyme.You have a plasmid with a concentration of 250 ng/µL, 10X reaction buffer, and your enzyme concentration is 20,000 units/mL.

How many µL of water, reaction buffer, DNA and enzyme would you combine to set up that reaction? Please show work

Solutions

Expert Solution

Total volume of reaction= 50 microliters

a) Given is 10X reaction buffer

Hence, to obtain 1X concentration, 10X buffer should be diluted 1: 10

C1V1=C2V2

10X X V1= 1X X 50 microliters

V1= 1 X 50/10= 5 microliters

5 microliters of 10X reaction buffer is added

b) Required DNA concentration is 1 microgram DNA/50 microliters

Given is 250 ng/microliters of plasmid DNA

1ng = 0.001 microgram

250 ng= 250 X 0.001= 0.25 microgram

Plasmid DNA= 0.25 microgram/ microliters

Amount of plasmid DNA added= 0.25 x 4= 1 microgram

Hence, 4 ml of 250 ng/ microliters of plasmid DNA is added to give a final concentration of 1 microgram of DNA in total volume of 50 microliters.

c) Given stock enzyme concentration is 20,000 units/ml

Required enzyme concentration is 10 units/50 microliters

Enzyme concentration in stock= 20000 units/ ml= 20000 units/ 1000 microliters = 20 units/ microliters

Hence, 0.5 microliters (10 units/ microliter) will be added to the total 50 microliters reaction volume to obtain 10 units/ microliters of enzyme

d) Distilled water= 50 microliters – (volume of enzyme + DNA + reaction buffer)

Distilled water= 50 –(5+4+0.5)= 50-9.5= 40.5 microliters

Final reaction of 50 ml:

5 microliters of 10X reaction buffer

4 microliters of 250 ng/ microliter plasmid DNA

0.5 microliters of 20000 units/ml enzyme

40.5 microliters of distilled water

gladiator answered 1 year ago

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