Restriction Enzyme Digest

Normal DNA digest with restriction enzymes is in a total volume of 50 ul (microliters.) You can do the digest in less vol. if, for example, you are only go to run it on a "minigel" (30ml agarose gel). In general digest 0.25 to 2 ug (micrograms) per digest.

Recipe

"X" ul DNA. 0.25 to 10 ul

5ul 10x buffer. Use the correct buffer - see chart

"Y"ul dH2O. Final volume equals 50ul

1ul ENZYME (if you add multiple enzymes, final volume of enzymes should not exceed 1% of total volume).

1ul of restriction enzyme always is greater than 1 unit. It is usually 10 to 20 units. 1 unit is enough enzyme to digest 1 ug DNA.

-Incubate 37oC for 1 to 3 hours.

-Sometimes you need to add Mg++ to the reaction (in the form of MgCl) because you add a large volume of DNA. That is, if "X" is large > 20ul, you need to add Mg. A good rule of thumb is for every 20ul of DNA add 1ul of 10mM MgCl.




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