Restriction enzyme digestion is a common technique used in gene cloning to prepare DNA fragments for insertion into a vector. Restriction enzymes recognize specific sequences in DNA and cleave the bonds between nucleotides at that site. The amount of restriction enzyme needed in microliters to completely digest one microgram of substrate DNA in one hour at the optimal temperature is defined as one unit of restriction enzyme activity. Restriction digestion is performed as part of gene cloning to isolate the desired DNA fragment, which is then inserted into a vector for cloning.
1. In The Name of God
Restriction Enzyme Digestion
in Gene Cloning
Hasan Ashoori
MSc student in Human Genetics
2. Introduction
A restriction digest is a procedure for preparing DNA for
analysis or other molecular purposes.
It is sometimes termed DNA fragmentation.
3. Enzyme
Digestion often done with Restriction Enzymes:
Restriction enzymes (or restriction endonucleases) are
bacterial enzymes
capable of cleaving double-stranded DNA.
8. Proper Use of Restriction Enzymes
*RE must be kept on ice and returned to ice
*Hold the tube by the center, do not hold the bottom of the
tube with your *warm* hands
*Check the pipette tip to be sure that enzyme is there! We
usually use 1uL of enzyme, and the enzyme solution is thick
(stored in glycerol).
9. A Unit of Restriction Enzyme Activity
The amount of restriction enzyme in microliters (uL) needed
to completely digest one microgram (ug) of substrate DNA in
one hour at the optimal temperature of the enzyme in a 50-uL
reaction volume.
10. A Unit of Restriction Enzyme Activity
The number of units of activity will help you:
determine how much of the enzyme needed in a
restriction digest.
too great of an excess may be detrimental to the digest.
Also, these enzymes are expensive and using more than
we need wastes money.
11. RE digestion in cloning
Restriction digest is most commonly used as part
of the process of the molecular cloning of
DNA fragment into a vector
12. Gene cloning
literature review
Gene Selection
Sequencing
Obtaining DNA
Bacteria Culture
DNA extraction
Gene amplification
PCR
Analyze PCR product
15. Protocol for PCR Product Digestion
Incubate tubes at 37oC for one hour.
16. Note that:
1-Because restriction digests are done for different outcomes, there are
no absolute rules and quantities.
2- Use at least the minimum number of Units necessary to cut the
DNA. Most people use more Units than absolutely necessary. This
speeds up the time needed and helps insure a complete digest.
3-Try to keep the total volume of restriction enzyme in the digest to
10% or less of the total digest volume. Thus if you have a 30 uL digest,
don't use more than 3 uL total of enzyme.
4-always add the enzyme last.
17. Extract Digested product
Using Low melt agar for extracting digested product
and eliminate additional fragments.
Low melting point agarose will facilitate DNA extraction
at later step, but regular agarose also works.
18. Recovery of DNA from Low Melting
Point Agarose Gels
Run digestion products on 0.7% LMP agarose gel in 1X TBE
Let solidify in cold room
Run gel in cold room, 100V
Cut out fragment of interest with clean razor blade and remove all
excess agarose from the DNA
19. Use long wave UV to visualize fragment
Melt gel slice at 65oC 5min.
Add buffer and Mix
Place at RT for 5min
20. Low Melt agar
Extract with equal vol phenol pH 7-8
Extract DNA by shaking vigorously for 2min.
Spin 10min. at RT
Precipitate with 1/10 vol 3M sodium acetate + 2vol
Ethanol
Wash with 70% ethanol
