The enzyme cut of DNA reacts to the heart.

, establish a standard enzyme-cutting reaction
Most researchers now follow a rule that 10 units of endo-cutting enzymes can cut 1 sg of
DNA from different sources and
. Typically, in a reaction system of 50 sl, an enzyme of 1 sl degrades 1 sg of purified DNA by reacting for 1 hour at the final concentration of 1X NEBuffer and at the corresponding temperature. If more enzymes are added, the reaction time can be reduced accordingly, and if the amount of enzymes is reduced, the corresponding extended reaction time (no more than 16 hours) can be fully reactive for many enzymes.
, the selection of the correct enzyme
it goes without saying that the selected enzyme must have at least one corresponding identification point on the substrate DNA. Enzymes that identify fewer bases cut substrates more frequently than enzymes with more bases. Assuming a DNA chain with a GC content of 50%, an enzyme that identifies 4 bases will cut an average of once per 44 (256) base, and an enzyme that identifies 6 bases will cut an average of one base every 46 (4096).
of the inner cut enzyme can be a sticky end (3' or 5' protruding end) or a flat end fragment. The adhesive end product can be connected to other compatible endase products, and all flat end products can be connected to each other. See the Compatible Cohesive Ends and Recleavable Blunt Ends article in the catalog.
, enzymes
cut enzymes should be placed on ice as soon as they are taken out of the refrigerator. Enzymes should be the last to be added to the reaction system (all other reactants should have been added and premixed before the enzymes were added). The amount of enzymes is based on the frequency of cutting on the substrate. For example, ULTRA-helix and encased DNA usually requires more than 1U/g of enzymes to be fully cut. See "Cutting Proton DNA" and "Cutting DNA by Enculation" in catalogs 244 and 264.
, DNA
DNA to be cut should have removed phenol, chloroform, ethanol,
EDTA
, detergent or excessive salt ion contamination, so as not to interfere with enzyme activity. Methylation of DNA should also be a factor to be taken into account in enzymatic cutting. For methylation, see methylation-related content on pages 252 to 253.
, buffer
for each enzyme NEB to provide the corresponding optimal buffer, can guarantee almost 100% enzyme activity. The buffer concentration should be 1X when used. Some enzymes require bSA of 100 mg/ml for optimal activity. In this case, we also provide 100X BSA (10mg/ml). Enzymes that do not require BSA will not be affected much if BSA is added. For more detailed information on buffers, see page 234.
6, reaction volume
endoenzyme vitality unit is defined as: within 1 hour, 50 μl reaction volume, the degradation of 1 μg of substrate DNA required enzymes for a unit of vitality. So enzyme: The proportion of DNA reaction can be determined from this. Smaller reaction volumes are more
by the error of
of the shifter. In order to control the concentration of glycelycerin below 5%, it is important to note that the volume of the enzyme does not exceed 10% of the total volume (general enzymes are stored in 50% glycelycester).
, hybrid
this is a very important but often overlooked step. To react fully, the reaction fluid must be fully mixed. We recommend mixing repeatedly with a gun, or by flicking the pipe wall with your finger, and then centrifuging quickly. Note: Non-oscillating!
, reaction temperature
the reaction temperature of most enzymes is 37 degrees C, and the internal cut enzyme isolated from the heatphilus requires a higher temperature. It generally varies from 50-65 degrees C. See page 244 of Activity of thermophiles at 37 degrees C. (Please refer to the catalogue!!) )
, reaction time,
1 enzyme living unit defined time of 1 hour. If more enzymes are added, the reaction time can be shortened accordingly, and if the amount of enzymes added is less, the reaction time can be extended to complete. See page 241 enzyme survival time in reaction.
, terminate the reaction
if the next enzyme cutting reaction is not carried out, the termination fluid can be used to terminate the reaction. At NEB we use the following reaction termination fluids: 50% glycelic, 50mM EDTA (pH8.0), and 0.05% bromophenol blue (10 sl/50 sl reaction fluid). If the next enzymatic reaction is to be performed, the reaction can be terminated by thermal inactive method (65 degrees C or 85 degrees C, 20 minutes).
thermal ineration is not suitable for all enzymes, see page 240 heat indation table for details. In addition, phenol/chloroform withdrawal can also be used to terminate the reaction.
enzymes should be stored
at -20 degrees C. A small number of enzymes must be preserved for a long period of time at -70 degrees C. For more information, please refer to the DATA SHEET of the relevant enzyme or the relevant section of the catalog. The 10X BUFFER and 100X BSA are stored at -20 degrees C. BSA cannot be mixed with NEBuffer and saved, otherwise BSA precipitation will occur.
, stability
have an active test of all enzymes every 1-2 months; Through more than 30 years of experience, we have found that most enzymes are very stable in the recommended preservation buffer at -20 degrees C. Stability will be reduced above -20 degrees C.
, control reaction
if you find that your DNA substrate can not be successfully cut open, you can conduct a controlled experiment to find out why. The method is as follows:
the substrate DNA (to-be-cut) without inchease is reacted simultaneously with the controlled DNA (there are several known enzyme cut points) that is added to the endochease. If the experimental results show that the substrate DNA degrades, the DNA is contaminated with
nucleic acid
enzymes in the purification process or reaction fluid;
if the test results show that the substrate DNA remains intact and the control DNA is successfully cut, the cause of the enzyme mass can be ruled out, at which point the control DNA and the DNA of the substrate to be cut can be mixed to react again to determine whether there are inhibitors in the sample. If inhibitors are present (usually salt, EDTA, or phenol), the control DNA in the mixture cannot be cut open.
.