Restriction Digestion   

Cat# BB-MTK040S (5 reactions) 
Cat# BB-MTK040 (10 reactions) 

Principle: A restriction enzyme or restriction endonuclease, or restrictase is an enzyme that cleaves double stranded DNA into fragments at or near specific recognition sites within that DNA molecule. These enzymes are found in bacteria and archaea and provide defense mechanism against invading viruses. Over 3,000 restriction enzymes are now known and more than 600 of these restriction enzymes are commercially    available for routine use in laboratories as a vital tool in molecular cloning for DNA modifications. Restriction enzymes are primarily used in the laboratory for generating 1) Sticky Ended and 2) Blunt Ended DNA   molecules for cloning purposes.

Isochizomers:

Any restriction endonuclease will cut only at a specific base sequence, no matter what DNA molecule it is acting on. However, a given Recognition sequence can be recognized by multiple enzymes called isochizomers e.g., Sma I and Xma I. These isochizomers can cleave the DNA at same or different position within the recognition sequence.

Factors affecting Restriction Enzyme Activity:

Buffer Systems: Tris-HCl is the most commonly used buffering agent in incubation mixtures, which is temperature dependent. Most restriction enzymes are active in the pH range 7.0 – 8.0.

Temperature: Most digestion reactions are carried out at 37°C. However, there are a few exceptions e.g., digestion with SmaI is carried out at lower temperatures (~25°C), while digestion with Taq I is carried out at higher temperature, i.e., 65°C.

Ionic Conditions: Mg²⁺ is an absolute requirement for all restriction endonucleases, but the requirement of other ions (Na⁺/K⁺) varies with different enzymes.

Methylation of DNA: Methylation of specific adenine orcytidine residues within the recognition sequence of the restriction enzyme affects the digestion of DNA.

Kit description: Using this kit, students will perform restriction enzyme digestion of the supplied plasmid DNA having 11,334 base pairs (bp) with two different enzymes, Hind III and Sal I. It has recognition sites for many restriction enzymes. Enzymes supplied in this kit, Sal I and Hind III have 2 and 7 recognition sequences on this supplied plasmid DNA. On Digestion with Sal I and Hind III, two and seven fragments of different sizes are obtained respectively. These bands are then resolved by Agarose gel electrophoresis to observe the different restriction enzyme patterns. Simultaneously, a molecular weight ladder will also be electrophoreses to assess the various fragment sizes. (Please see the agarose gel electrophoresis profile of the digested DNA at the last page).

Materials Provided (For 5 Reactions only): The list below provides information about the materials supplied in the kit. The products should be stored as suggested. Use the kit within 3 months of arrival.

Procedure:

Setting up the Restriction Digestion Reaction:

• Place the vials containing restriction enzyme (Hind III and Sal I) on ice.
• Thaw the vials containing substrate (Plasmid DNA) and Reaction buffer.
• Prepare two different reaction mixtures using the following

Reaction 1 (Hind III digestion)

Reaction 2 (Sal I digestion)

• Incubate the vial at 37°C for 2
• Meanwhile, prepare a 7% agarose gel for electrophoresis. (Please follow the direction at the last page).
• After 2 hours add 5 μl of gel loading Dye to each
• Load the digested samples, 5 μl of Control DNA and 5 μl of DNA Ladder, note down the order of
• Electrophoreses the samples at 50-100 V for 1-2hours.

Observation:
Preparation of 0.7% Agarose gel

 electrophoresis:

• Prepare 7% agarose solution in 50 X TAE buffer provided (e.g. for 50 ml of agarose gel add 0.35 gm of agarose, 1 ml of TAE & 49 ml of sterile water).
• Boil until the agarose is completely
• After boiling let the to be cooled down around 37⁰C – 40⁰C.
• Add 5 µl of Ethidium Bromide from the supplied stock (very carefully) & mix properly.

• Seal the gel casting tray on two sides; place the comb in the gel tray in appropriate
• Pour the agarose mixture in the tray containing
• Let the agarose to be solidified in the Remove the seal from the two sides without disturbing the gel.

• Next keeps the gel tray in the tank containing 1X TAE Keep the wells of the gel in the cathode (Negative) side. The buffer level in the tank should be maintained above the gel tray.
• Lift the comb from the gel gently to avoid damaging of wells; the gel is now ready for
• Connect the cords of electrophoresis tank with the power pack before loading the
• To prepare samples for electrophoresis add 3-5 µl of 6X gel loading dye into the sample & mix well by pipetting or pulse Load 20 µl of sample into the well.
• Then switch on the power pack & adjust the voltage in between 60V to
• Continue the electrophoresis until the dye reaches to1/3^rd or above of the gel.

Adapted from internet