Restriction Digestions You need to digest 100ng of your plasmid DNA with BamHI.

Question

Restriction Digestions You need to digest 100ng of your plasmid DNA with BamHI. The maximum volume of your sample that you can load on the agarose gel is 30 Your DNA sample is 20ug/ml. This has been a particularly difficult DNA sample to digest, so you want to use the maximum The enzyme is in 50% glycerol and the final concentration of glycerol in the reaction cannot be greater than 5%. The Gel Sample Buffer is a 3X stock. Keep this in mind since it must be included in your 30? final volume. amount of enzyme in this reaction. To assemble this reaction with 100ng DNA and the maximum permissible volume of enzyme, you would combine the following: ul DNA ?110X Reaction Buffer water 1 Hindillrestriction enzyme After 60 minutes at 37°C, add ?13X Gel Sample Buffer

Explanation / Answer

5?l DNA

9?l water

3?l 10X reaction buffer

3?l HIndII restriction enzyme

10?l gel sample buffer

DNA concentration is 20µg/ ml

thus 20 X1000 ng is present in 1000µl

       100ng DNA is present in 1000 X 100/ 20000= 5µl.

we need to take 5µl DNA.

reaction buffer concentration is 10 X, so we need to dilue to it 1X .or 10fold dilution.

As the final volume should be 30µl, thus we will require : 30/10= 3µl of reaction buffer

Gel sample buffer is 3X concentrated, working concentration should be 1X, thus it shoud be 3 fold diluted.

for 30µl final volume we will equire 30/3= 10µl of gel sample buffer.

HIndIII resiriction enzyme is in 50% glycerol. As the final concentration of glycerol can not be more than 5%, so its required to dilute 50/5 = 10 fold. Thus for 30µl final volume, we will require 30/10= 3µl of HIndIII restrction enzyme.

the rest volume is need to make up by water, so that the total volume will be 30µl

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