2024年4月23日发(作者：微信恢复好友)

限制性内切酶酶切的常见问题及解决方法（The common problems and

solutions of restriction endonuclease digestion）

The common problems and solutions of restriction endonuclease

digestion

Restriction enzyme digestion of the common problems and

solutions, personally feel very well summed up, specially

posted for you to share.

Enzyme digestion problems, see enzyme instructions, the

corresponding reagent company directory. The enzymes produced

by different companies, the source of strains, the preparation

process, purity, viability and enzymatic activity may be

optimized differently,.

Can be found in the above definition, unit of enzyme

preservation conditions, enzyme system of [buffer and other

enzymes such as buffer, double cut enzyme reaction temperature

[some enzymes in 50, 55 or 30 temperature reaction, whether

methylation affects the enzyme activity and whether there is

an asterisk, asterisk may appear live factors and protective

base, isocaudarner, isoschizomer

1. enzymes can not be cut or incomplete

The 1.1 plasmid is the most common or poorly expressed inhibitor.

The presence of miscellaneous proteins can affect enzyme

digestion, showing A260/A280 below 1.8; inhibitors are common

in phenols, salts, and ethanol; [re DNA, using reliable kits

or reliable manual extraction reagents,

1.2 enzyme problems: make sure the enzyme is effective

[although many enzymes have expired time, but expired as long

as the enzyme can be effectively cut, available. Make sure the

enzyme is not effective. Mark it and replace it.

[note] topic: by endonuclease

A. endonuclease, if no special requirement, keep -20~-30

degrees. Is not as low as possible, the enzyme is usually

preserved in 50% glycerol buffer, when the temperature is too

low, will freeze (enzyme transport process exception, because

the enzyme requires low temperature transport, so the

convenient situation is the use of dry ice, the enzyme will

freeze, but the freezing times is limited, is a relatively

better choice), if is often used, the enzyme will be repeated

freezing and thawing, thereby reducing the activity. Of course,

if the temperature is too high, ha ha, you want to go.

B. enzyme should be used in ice box for enzyme extraction. It's

clear to everyone, but it's no harm putting too much emphasis

on it. Some of the students because the lab is put out, the

enzyme in the ice box, but there are two ignored: when taking

the enzyme, the hand does not hold the upper end of the pipe,

and the grip at the bottom, equivalent to hand in to the enzyme

by heating; some enzymes are placed in the ice box, but the

absorption of the enzyme when will the enzyme take out. Once

or twice a day, it can affect enzyme activity.

C. enzyme should be tightened as soon as possible after the lid.

Sometimes we can find that even if -20~-30 degrees are placed,