Restriction Enzyme Digestion
Aim: To digest the pUC18 DNA with BamH1 enzyme
Principle:
Restriction endonucleases are the class of enzymes that are used to cleave DNA at specific sites called Restriction sites. Every restriction enzyme has a specific restriction site at which it cuts a DNA molecule. For example restriction sequence for BamHI is GGATCC (type II restriction enzyme. The most abundantly used restriction enzymes are type II restriction enzymes which cleave at specific restriction site only. These endonucleases function adequately at pH 7.4 but different enzymes vary in their requirements for ionic strength usually provided by sodium chloride and magnesium chloride. It is also advisable to add a reducing agent such as dithiothreitol (DTT) which stabilizes the enzymes and prevents their inactivation. Any variation in the concentration of Na or Mg can lead to changes in specificity of the enzyme so that it can cleave at additional or nonstandard restriction sequences. The phosphodiester bond is cleaved between specific bases, one on each DNA strand, no matter the source of the DNA. The restriction endonucleases produce either sticky or blunt ends upon cleavage. Also based on the number of sequences identified for cleavage they can be tetracutter (4), hexacopter (6) or octacutter (8).
Materials Required For Restriction Enzyme Digestion
- pUC18 DNA
- BamH1 enzyme
- 10X buffer
- 1Kb Ladder
- Sterile water
- Agarose
- 6X loading dye
- 1.5 ml Sterile Vials
- Ethidium Bromide
- 1X TAE buffer
Procedure:
- Take 1.5 μg of PUC18 DNA (10 ul) in a fresh Eppendorf.
- To this, add 11.5 µl of sterile water followed by 5 µl of 10X buffer.
- Add 1.5 μl of BamH1 enzyme (1 unit) and incubate the mixture at 37˚C for 2 hrs.
- Prepare 0. 7% agarose gel and load the samples including 1 Kb DNA ladder, undigested pUC18 DNA and BamH1 digested PUC18 DNA.
- Run the gel at 100 V for 1 hr.
- Visualize the gel under UV illuminator.
- 10ul of the sample and 2ul of the dye were mixed
- Load 10ul of this into the gel
Reaction Protocol:
PUC18 DNA: 10 µl (1.5ug)
Sterile water: 11.5 µl
10X buffer: 2.5 µl
BamH1: 1 µl (1ug)
‐‐‐‐‐‐‐‐‐‐
Total: 25 µl (Incubate at 37˚ C for 1‐2 hrs)
Results and Discussion: (make it appropriate for the DNA and restriction enzyme used in your study)
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