30
KAI-OLAF NETZER
Four variables have to be known:
• the number of restriction enzyme sites in Iambda phage DNA,
• X, the amount of cloned DNA to be cut in )..lg,
• the number of sites within the cloned DNA, and
• Y, the length of the cloned DNA fragment.
Example: A digestion is tobe set up using an enzyme with 6 recognition sites
in Iambda. 1 )..lg plasmid DNA shall be digested. The fragment contains two
sites forthe enzyme, and is 1000 bp long. 16 U ofenzymewould be needed to
completely digest the DNA in one hour. If more time, e.g. three hours, is
allowed, the amount of enzyme may be reduced to approximately one third,
5 U. If preparations of genomic DNA aretobe digested, a good rule ofthumb
is to add 3 U per )..lg DNA, and to allow very long incubation tim es of several
hours to over night (16 hours). Ifthe DNA is impure and contains contaminating protein, it may be necessary to add 1-2 U/)..lg of fresh enzyme after
several hours and to incubate an additional three to five hours.
Materials
Supplies
• Heating block or water bath (temperature range 37 to 65°C)
• 0.5 or 1.7 ml reaction tubes
Reagents and
solutions
• Micropipettes, 1-10 )..ll and 10-100 )..ll
• Autoclaved pipet tips
• DNA, 0.1-0.2 )..lgl)..ll in water orTE buffer (10 mM Tris-HCl pH8.0, 1 mM
EDTA)
• Enzyme (should not exceed 1/10 of the total volume)
• Buffer (usually supplied as lOx reaction buffer by the manufacturer. It
may contain NaC110-150 mM, Tris-HCl pH7.5-8.0 50-150 mM, MgCb 010 mM, KCl, and DTT in varying concentrations depending on the enzyme and manufacturer)
• Ultrapure water
• Acetylated bovine serum albumin (BSA, Sigma)
KAI-OLAF NETZER
Four variables have to be known:
• the number of restriction enzyme sites in Iambda phage DNA,
• X, the amount of cloned DNA to be cut in )..lg,
• the number of sites within the cloned DNA, and
• Y, the length of the cloned DNA fragment.
Example: A digestion is tobe set up using an enzyme with 6 recognition sites
in Iambda. 1 )..lg plasmid DNA shall be digested. The fragment contains two
sites forthe enzyme, and is 1000 bp long. 16 U ofenzymewould be needed to
completely digest the DNA in one hour. If more time, e.g. three hours, is
allowed, the amount of enzyme may be reduced to approximately one third,
5 U. If preparations of genomic DNA aretobe digested, a good rule ofthumb
is to add 3 U per )..lg DNA, and to allow very long incubation tim es of several
hours to over night (16 hours). Ifthe DNA is impure and contains contaminating protein, it may be necessary to add 1-2 U/)..lg of fresh enzyme after
several hours and to incubate an additional three to five hours.
Materials
Supplies
• Heating block or water bath (temperature range 37 to 65°C)
• 0.5 or 1.7 ml reaction tubes
Reagents and
solutions
• Micropipettes, 1-10 )..ll and 10-100 )..ll
• Autoclaved pipet tips
• DNA, 0.1-0.2 )..lgl)..ll in water orTE buffer (10 mM Tris-HCl pH8.0, 1 mM
EDTA)
• Enzyme (should not exceed 1/10 of the total volume)
• Buffer (usually supplied as lOx reaction buffer by the manufacturer. It
may contain NaC110-150 mM, Tris-HCl pH7.5-8.0 50-150 mM, MgCb 010 mM, KCl, and DTT in varying concentrations depending on the enzyme and manufacturer)
• Ultrapure water
• Acetylated bovine serum albumin (BSA, Sigma)