17 Genomic Libraries
269
Procedure
Partial enzyme digestion
1. Transfer 100 Jlg of genomic DNA {0.1-0.5 mglml) to a microcentrifuge
tube. Dilute to 900 J.!l and add 100 J.!l of the appropriate 10 x restriction
enzyme buffer. Remove a 40 J.!l aliquot, add to 10 J.!l of stop solution, and
place on ice. Incubate the remainder at 37°C for 10 minutes.
2. Add to the main reaction mix sufficient enzyme to digest completely only
20% of the DNA in 60 minutes. Remove 40 J.!l aliquots every 10 minutes
and add each to 10 J.!l of stop solution as in Step 1. Follow this procedure
for 90 minutes.
3. Electrophorese the aliquots of digested DNA on a O.So/o agarose gel, being
certain to include markersthat span a range of sizes from 10 kb- 100 kb.
Determine the time interval that yielded the highest concentration of
fragments that equals the optimal insert size range of the selected vector.
Between 10 and 20% of the total amount of DNA should be within this
size range. One may need to repeat the experiment if the previous results
were unsatisfactory, adjusting the enzyme concentration accordingly
(usually 10 x more or 10 x less).
4. Once suitable conditions are determined, scale up the reaction to 1 mg of
DNA in a 10 ml final reaction volume. Remove 3.3 ml of the digest 5
minutes before the optimal time point, at the optimal time point and
5 minutes after. Combine the aliquots, add to 10 mls phenol-chloroform,
and mix well. Separate the phases by centrifugation at 5000- 10000 x g for
5 minutes. Remove the supernatant and safely dispose of the organic
phase. Add 1 ml of 5 MN aCl and 30 ml100o/o ethanol to the supernatant,
chill in a dry ice/ethanol slurry for at least 5 minutes, and centrifuge at
20,000 rpm. Dilute the precipitated DNA in 1 ml STE buffer {10 mM TrisCl (pH 7.5), 10mMNaCl, 1 mMEDTA (pH 8.0)) andcheck 10J.!lona0.5o/o
agarose gel.
Size fractionation
Sucrose density gradient centrifugation for fractionating DNA according to
size. This technique is also used to purifythe arms oflambda bacteriophage.
1. Dissolve the digested DNA in STE bufferat a concentration of 1 mglml.
269
Procedure
Partial enzyme digestion
1. Transfer 100 Jlg of genomic DNA {0.1-0.5 mglml) to a microcentrifuge
tube. Dilute to 900 J.!l and add 100 J.!l of the appropriate 10 x restriction
enzyme buffer. Remove a 40 J.!l aliquot, add to 10 J.!l of stop solution, and
place on ice. Incubate the remainder at 37°C for 10 minutes.
2. Add to the main reaction mix sufficient enzyme to digest completely only
20% of the DNA in 60 minutes. Remove 40 J.!l aliquots every 10 minutes
and add each to 10 J.!l of stop solution as in Step 1. Follow this procedure
for 90 minutes.
3. Electrophorese the aliquots of digested DNA on a O.So/o agarose gel, being
certain to include markersthat span a range of sizes from 10 kb- 100 kb.
Determine the time interval that yielded the highest concentration of
fragments that equals the optimal insert size range of the selected vector.
Between 10 and 20% of the total amount of DNA should be within this
size range. One may need to repeat the experiment if the previous results
were unsatisfactory, adjusting the enzyme concentration accordingly
(usually 10 x more or 10 x less).
4. Once suitable conditions are determined, scale up the reaction to 1 mg of
DNA in a 10 ml final reaction volume. Remove 3.3 ml of the digest 5
minutes before the optimal time point, at the optimal time point and
5 minutes after. Combine the aliquots, add to 10 mls phenol-chloroform,
and mix well. Separate the phases by centrifugation at 5000- 10000 x g for
5 minutes. Remove the supernatant and safely dispose of the organic
phase. Add 1 ml of 5 MN aCl and 30 ml100o/o ethanol to the supernatant,
chill in a dry ice/ethanol slurry for at least 5 minutes, and centrifuge at
20,000 rpm. Dilute the precipitated DNA in 1 ml STE buffer {10 mM TrisCl (pH 7.5), 10mMNaCl, 1 mMEDTA (pH 8.0)) andcheck 10J.!lona0.5o/o
agarose gel.
Size fractionation
Sucrose density gradient centrifugation for fractionating DNA according to
size. This technique is also used to purifythe arms oflambda bacteriophage.
1. Dissolve the digested DNA in STE bufferat a concentration of 1 mglml.