Digestion Tips
These are some Tips about digestions I read the other day.
Things that make your digestion fail:
- Extremely high concentration of enzyme
- Prolonged incubation time with enzyme
- 10 units of restriction enzyme is sufficient to overcome variability in source, purity, quantity of DNA
- Keep glycerol concentration at les than 5% in a reaction (The restriction enzymes are provided in 50% glycerol ==> that is why they don't freeze) hence the restriction enzyme should NOT exceed 10% of the total reaction volume
- Mixing, "ficle" the reaction tube and follow qith a quick spin-down in the microcentrifuge.
- DNA must be free of contaminants (phenols, chloroform, alcohol, EDTA, detergents, salts)
- Control reactions
a) Incubate experimental DNA without restriction enzyme (degradation indicates contamination in DNA or buffer)
b) Use control DNA for the reaction with restriction enzyme. Control DNA has multiple known sites for the enzyme (e.g. lambda DNA)
if a) is not cleaved and b) is, you can mix them to assess presence of inhibitors (EDTA, salts...) ==> the control DNA won't cut.
How is ONE unit of restriction enzyme defined?
It's the enzyme activity that cleaves 1μL DNA suspended in 50μL buffer in 60 minutes at the appropiate temperature.
How can I calculate the optimum number of enzyme units for my restriction digest?
Reference DNA is used, you know the number of cleavage sites for the enzyme you are using.
e.g. EcoRI 1 unit cleaves 1μg λ DNA in 1 hour at 37°
λ DNA has 48,502 bp it has 6 cleavage sites
1 unit cleaves DNA with an average cleavage-site frequency of 1/8084bp
If I have a plasmid of Xbp carrying Y enzyme sites
1) I check the site frequency
2) I calculate, if I need 1 unit of enzme for a frequency o 1/8084 (EcoRI), how many units do I need for my plasmid frequency? This will usually be low, which is why we normaly use 10 units.
