(10 points) Your current project involves cloning the onlyatnight gene from vamp

Question

(10 points) Your current project involves cloning the onlyatnight gene from vampue bats. You have isolated the genomic DNA and must now digest it with two restriction enzymes. More specifically, you need to digest 5ug of the genomic DNA with both Sall and HindIII restriction enzymes. The maximum volume of your sample that you can load on the agarose gel is 60ul. Your genomic DNA sample is 250?g/ml. This has been a particularly difficult DNA sample to digest with Sall, so you want to use the maximum amount of SalI enzyme in this reaction. The enzyme is in 50% glycerol and the final concentration of glycerol in the reaction cannot be greater than 5%. The HindIII enzyme has already been diluted into 1XReaction Buffer to a final concentration of 0.50 Units/?| (ignore any possible glycerol in the diluted HindIII enzyme). You need to add 10 Units HindIII enzyme. The Gel Sample Buffer is a 6X stock. Keep this in mind since it must be included in your 60 l final volume. Express your answers to the nearest 0.11. To assemble this reaction, you would combine the following: ?? DNA ?1 10X Reaction Buffer,? th od I water ?1 Sall restriction enzyme ul HindIII restriction enzyme X After 60 minutes at 37°C, add ?16X Gel Sample Buffer × ?

Explanation / Answer

Given genomic DNA sample = 250 µg/ml

requirement = 5 µg

Total reaction volume = 60 microlitre

so for 5 microgram of DNA

250 X x = 60*5

x = 300/250 = 1.2 microlitre

required Hind III enzyme = 10units

and we have a0.50units /microlitre

so the required volume of Hind III = 20 microlitre…………………(0.5U in 1microlitre , so by unitary method, in 20 microlitre it will be 10U)

10X reaction buffer will be made to 1X by diluting it,

so 10X x X = 1X x 60

X = 6microlitre

The Sal I enzyme is in 50% glycerol and the maximum glycerol can be 5%

So, it need to be diluted up to 10times.

it means in a reaction of 100 microlitre max 5 microlitre SalI can be added, so, for 60 microlotre the vol. required = 3 microlitre.

Sample buffer = 6X

so for 60microlitre 1X the volume required will be

= 6X x X = 1X *60

X = 10microlitre

So, for a reaction of 60 microlitre the reaction will be made as fillows:

DNA = 1.2 microlitre

Sample reaction buffer = 6 microlitre

Hind III = 20 microlitre

6X gel sample buffer = 10 microlitre

Sal I = 3 microlitre

water will be added to make up volume to 60 microltre

So, 19.8 microlotre water will be added in the reaction.

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