1. In The Name of GodRestriction Enzyme Digestion in Gene Cloning Hasan Ashoori MSc student in Human Genetics

2. IntroductionA 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. 4. recognition sequence?? Restriction enzymes recognize specific sequences in DNA and then cleave the bonds between thenucleotides at that site. 5. Schematic picture of RE 6. www.rebase.neb.com 7. 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). 8. A Unit of Restriction Enzyme ActivityThe 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-uLreaction volume. 9. A Unit of Restriction Enzyme ActivityThe 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. 10. 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 11. Gene cloningliterature review Gene Selection Sequencing Obtaining DNA Bacteria CultureDNA extractionGene amplificationPCRAnalyze PCR product 12. RE DigestionPCR productVector 13. Gene cloning in a video clip 14. Protocol for PCR Product DigestionIncubate tubes at 37oC for one hour. 15. 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. 16. Extract Digested productUsing 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. 17. 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 18. Use long wave UV to visualize fragment Melt gel slice at 65oC 5min. Add buffer and Mix Place at RT for 5min 19. 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 20. Digestion problems 21. Cloning without RE! 22. Thanks for your attention