Molecular Biology Topics

RESTRICTION ENZYME (ENDONUCLEASE) DIGESTION

Written by MicroDok

The digestion of restriction enzyme (endonuclease) is explained in this section


Aim of the experiment
: To digest the pUC18 DNA with BamH1 enzyme

PRINCIPLE FOR RESTRICTION ENZYME DIGESTION EXPERIMENT


Restriction endonucleases (aka, restriction enzymes or endonucleases) are the class of enzymes (e.g., BamH1) 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 enzyme so that it can cleave at additional or non‐
standard 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), hexacutter (6) or
octacutter (8).

Illustration of experiment showing restriction enzyme at work

MATERIALS REQUIRED FOR RESTRICTION ENZYME DIGESTION

  • pUC18 DNA
  • BamH1 enzyme
  • 10X buffer
  • 1Kb Ladder
  • Sterile water
  • Agarose
  • 6X loading dye
  • 5 ml Sterile Vials
  • Ethidium Bromide
  • 1X TAE buffer

PROCEDURE FOR RESTRICTION ENZYME DIGESTION

 

  • 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 units) 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
  • Laod 10ul of this in to 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:

 

References:  DEPARTMENT OF BIOTECHNOLOGY, SCHOOL OF BIOENGINEERING, SRM UNIVERSITY

 

About the author

MicroDok

Leave a Comment