Enzymatic digestion of DNA fragments

Summary

Restriction endonucleases are a class of DNA hydrolases that recognize specific nucleotide sequences in double-stranded DNA molecules, and the discovery and application of these enzymes can be used in DNA recombination-based bioengineering technologies.

Operation method

Enzymatic digestion of DNA fragments

Principle

Enzymatic cleavage refers to the use of sticky end ligation must be performed on the target DNA molecule and the vector molecule in order to obtain the appropriate sticky ends for ligation.

Materials and Instruments

DNA fragments
TE Buffer
Centrifuge Constant Temperature Bath Liquid Extractor Electrophoresis Instrument Electrophoresis Tank UV Viewer

Move

I. EcoR Ⅰ, Hind Ⅲ single enzyme digestion and double enzyme digestion
1. Take 2 clean, sterilized new Eppendof tubes and add to each according to the table below.

	A (μl)	B (μl)
Sterilized water	13	13
EcoR Ⅰ buffer	2	0
Hind III buffer	0	2
λDNA (or plasmid DNA)	4	4
EcoR Ⅰ	1	0
Hind III	0	1
Total volume	20	20

2. Hold at 37°C for 1-4 hours.
3. 65-70°C for 10 min after the end of holding time to inactivate the enzyme (or extract with chloroform if it is a heat-stable enzyme).
4. Take about 2 μl of the reaction solution and add 2 μl of electrophoresis addition buffer to it.Second, EcoR Ⅰ, Hind III double enzyme digestion
1. Take a clean, sterilized Eppendof tube and add the reagents according to the table below.

Add reagent	Volume (μl)
Sterilized water	12
MULT buffer	2
λDNA (or plasmid DNA)	4
EcoR Ⅰ	1
Hind III	1
Total volume	20

2. Hold at 37°C for 1-2 hours.
3. 65-70°C for 10 min after the end of holding time to inactivate the enzyme (or extract with chloroform if it is a heat-stable enzyme).
4. Take about 2 μl of the reaction solution and add 2 μl of electrophoresis addition buffer to it.

Caveat

1. The order of sample addition is water, buffer, DNA and finally enzyme, and should not be reversed.

2. The enzyme addition step should be carried out in an ice bath, and the water, buffer and DNA to be cut should be mixed well before adding the enzyme.

3. The amount of water in the reaction volume should be as small as possible, i.e. the volume of the reaction system should be as small as possible. Generally, when hydrolyzing 0.2~1 ug of template DNA, the volume should be controlled within 20~30 ul. But make sure that the volume of added enzyme is not higher than 1/10 of the total volume, because the restriction endonuclease is preserved in 50% glycerol, if the volume of added enzyme is higher than 1/10 of the total volume, the concentration of glycerol in the reaction solution will be more than 5%, and this concentration will inhibit the endonuclease activity.

4. In order to control the reaction volume and promote the reaction, the template DNA concentration should be very high, otherwise the reaction system DNA concentration is too low will cause the enzyme reaction kinetics change, reduce the effect of digestion, this time have to increase the reaction volume; and generally in order to increase the stability of the DNA storage, DNA is more preserved in TE buffer, such as the reaction system is too much to add the template DNA solution is bound to If too much template DNA solution is added to the reaction system, it will inevitably cause the EDTA concentration in the reaction system to increase, and inhibit the enzyme. Therefore, if the concentration of substrate DNA is too low, it should be concentrated.

Common Problems

The operation of single enzyme cleavage is relatively simple, but double enzyme cleavage if the buffer used for the two enzymes has different compositions (mainly different salt ion concentrations) or different reaction temperatures, then the following measures can be used to solve the problem: 1) use one enzyme for cleavage, then ethanol precipitation to recover the DNA molecules and then use the other enzyme for cleavage; 2) carry out the cleavage of the enzyme with the low-salt requirement first, and then add the salt ion concentration to the reaction requirement of the enzyme with the high salt and add the the second enzyme for digestion; 3) double digestion using a universal buffer. The specifics should be carried out according to the reaction requirements of the enzyme to avoid the loss of vigor as much as possible.

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