DNA's restrictive endoenzyme cutting reaction.

experiment seven DNArestrictive endoenzyme cutting reactionExperimental Purposethrough this experiment to learn the basic principles and experimental techniques of DNA's restrictive endoenzyme cutting reaction.
. ( Experimental Principle ) 1. Restrictive endoenzymes can be specifically bind to a specific site within or near a DNA sequence called a restrictive enzyme recognition sequence, and double-stranded DNA is cut. It can be divided into three categories: class I. and class III. Enzymes in the same
protein
molecules have both cutting and modification (methylation) and rely on the presence of ATP. I. Sterase binds to DNA that identifies the bit and randomly cuts the identification bit not far away, while III. Classase cuts the DNA molecule at the identification point and then dissophases it from the substrate. Class II. Class consists of two enzymes: a restrictive kernel nuclease (restriction enzyme), which cuts a specific nucleotide sequence; The other is a separate methylase, which modifies the same identification sequence. The vast majority of Class II. Class limitases recognize echo symmetrical specific nucleotide sequences with a length of 4 to 6 nucleotides (e.g. EcoRI. identify six nucleotide sequences: 5"-G-AATTC-3"), and a few enzymes identify longer sequences or simple sequences. II. In the identification sequence, some of the steroid cutting points are cut at the symmetrical axis to produce flat-end DNA fragments (e.g. Sma:5"-CCC-GGG-3"), and some cutting bits are on the symmetrical axis side, resulting in DNA fragments with single-stranded protruding ends called sticky non-ends, such as EcoRI. 5"... G-AATTC... 3" →5"... G AATTC... 3" ;3"... CTTAA-G ... 5" →3"... CTTAA G... 5"

2.DNA purity, buffer, temperature conditions, and restrictive endoenzymes themselves can affect the activity of restrictive endoenzymes. Most restrictive endoenzymes are not affected by RNA or single-stranded DNA. When trace amounts of contaminants enter the restricted endoenzyme storage fluid, it affects its further use, so a new straw head is used each time the restrictive endoenzyme is drawn. If two restrictive endoenzymes are used, it is important to pay attention to providing their respective most suitable salt concentrations. If the same buffer is available for both, it can be hydrolysed at the same time. If different salt concentrations are required, the restricted endoenzyme at the low salt concentration must first be used, then the salt concentration must be adjusted, and then hydrolyzed with the restrictive endoenzyme at the high salt concentration. It is also possible to use iso-volume phenol/chloroform pumping, plus 0.1x volume 3mol/LNaAc and 2x volume aqueous ethanol, mixed and placed in a low-temperature refrigerator at -70c for 30 minutes, centrifugal,
driding
and re-dissolved in the buffer for a second enzymatic cutting reaction.

. 3. DNA restrictive endoenzyme cutting map, also known as the physical map of DNA, it consists of a series of location-determined a variety of restrictive endoenzyme cutting points, expressed in a straight line or ring chart. There are many ways to construct a DNA restrictive endoenzyme map. A combination of a variety of restrictive endoenzymes is often used to determine the enzyme cut points and their relative positions of various enzymes by comprehensively analyzing the size of the restricted fragments obtained by simultaneous cutting of multiple enzymes and different combinations of enzymes. The value of the use of an enzyme cut map depends on its accuracy and accuracy. In the process of enzymatic map making, in order to obtain a clear electrophoresis map, the general DNA dosage is about 0.5-1 μg. The enzyme reaction of restrictive endoenzymes varies most appropriately, with various enzymes having their corresponding enzyme-cutting buffers and the most suitable reaction temperature (most of which are 37 degrees C). For the proton DNA enzymatic process, the limiting endoenzyme dosing can be digested for 1-2 hours according to the standard system of 1 sg DNA plus 1 unit enzyme. However, to fully enzymatic must increase the amount of enzymes, generally increased by 2-3 times, or even more, the reaction time should be appropriately extended.
.experimental instruments and equipment )horizontal electrophoresis device, electrophoresis device, desktop high-speed
centrifuge
,
thermostat
water bath pot, micro-liquid gun, microwave oven, ultraviolet transmission device, photographic systemexperimental materialzirconate DNA; recombinant pBS material or pUC19 granules; EcoRI enzymes and their enzyme-cut buffers; Hind III. Enzymes and their enzyme-cut buffers;
agar
(Agarose).
. Preparation
the
:
1, 5×TBE electrophoresis buffer: see experiment II.

2, 6 × electrophoresis sample buffer: see experiment II.

3, ethyl bromide ingots: see experiment II.
. ( experimental step ) 1. Number the clean, dried and sterilized eppendorf tube (preferably 0.5 ml) and add DNA 1 sg and the corresponding restrictive endoenzyme reaction 10× The buffer is 2 sl, and then the total volume is 19 sl, the solution in the tube is mixed and then added 1 sl enzyme solution, the solution is mixed with a finger flick of the tube wall, can also be shaken with a trace centrifuge, so that the solution is concentrated at the bottom of the tube.

. 2. After mixing the reaction system, place the eppendorf tube on a suitable support (e.g. plugged into a foam plate) and keep the water bath warm for 2-3 hours at 37 degrees C, so that the enzyme cut reaction is complete.

. 3. Add 2 μl 0.1mol/L
EDTA
(pH8.0) to each tube and mix well to stop the reaction and place in the refrigerator to store the spare.

. 4. Preparation of agarose gel analysis endoenzyme cutting reaction resultspreparation of 1.5% agarose gel, take 10 sl enzyme lysate and 2 sl 6× carrier liquid mixed electrophoresis. Hold the voltage at 60-80V, the current at 40mA. After electrophoresis, with EB dyeing 20-25min, UV lamp observation results.
. Based onobservations
UV lamp, the electrophoresis schematic is drawn and the matters to be noted in the endoenzyme cutting reaction are discussed.
. Thearrangement )
6 hours1 day can be completed, in the morning to do endoenzyme cutting reaction, in the afternoon to do electrophoresis testing.
.