Restriction Enzymes are also known as Molecular scissors. Produce cohesive/sticky and blunt ends. Many restriction enzymes are present have their own particular Restriction sites (Palindromic Sequences)

1. 12-DEC-2018
Practical# 7
RestrictionOf BacterialPlasmidby Restriction
Enzymes
Introduction:
Restriction Digestion is the process of cutting DNA
molecules into smallerpieces with special enzymes
called Restriction Endonucleases(sometimes just called
Restriction Enzymes or RE's). Because of this property
the restriction enzymes are also known as molecular
scissors. The restriction enzymes are named from the
cellularstrainfrom which they are isolated.Restriction
Enzymes are delicate and need to be treated carefully.
Because enzymes are proteinsand proteins denature as
the temperature is increased,RE's are always stored in a
freezer until they are used.
Principle:
Restriction enzymes recognize specific sequences in the
double stranded DNA molecule (for example GATATC)
and then cut the DNA to produce fragments, called
restriction fragments. The target site or sequencewhich the
restriction enzyme recognizes is generally from 4 to 6 base
pairs and arranged in a palindromic sequence. Once it is

2. located, theenzyme will attachto the DNA molecule and
cut each strand of the doublehelix. The restriction enzyme
will continue to do this along the full length of the DNA
molecule which will then break into fragments. The size
of these fragments is measured in base pairs or kilobase
pairs (1000 bases).
 Sticky ends: enzymes that leave single-stranded
overhangs are said to produce sticky ends. Sticky
ends are helpful in cloning because they hold two
pieces of DNA together so they can be linked by
DNA ligase.
 Blunt ends: Enzymes which cut straight down the
middle of a target sequenceand leave no
overhang. They are harder to ligate together (the
ligation reaction is less efficient and more likely to
fail) because there are no single-stranded
overhangs to hold the DNA moleculesin position.

3. Requirements:
In this experiment Plasmid DNA is digested with two
restriction enzymes; EcoRI and HindIII. - Lambda (λ)
Plasmid DNA – 5.0 µl ,10XAssayBuffer of EcoRI – 2.5 µl ,
Milli Q water –16.5 µl , EcoRI – 1.0 µl.
- PLasmid DNA – 5.0 µl, 10X AssayBuffer of HindIII – 2.5
µl , Milli Q water –16.5 µl , HindIII – 1.0 µl.
Procedure:
1.Before starting the experiment, crush ice and place the
vials containingLambda DNA, Restriction Enzymes and
Assay Buffers onto it.
2.After preparing the two reaction tubes, mix the
componentsby gentle pipettingand tapping. 3.Incubate
the tubes at 37◦
C for 1 hour.
4. After 1 hourincubation, immediately place the vials at
room temperature(15-25 ◦
C) for 10 minutes. 5.. Run the
samples on agarose gel.

4. 6.After Agarose Gel Electrophoresis. Visualize the DNA
bands using UV Transilluminator.
7. After runningthe digested samples on agarose gel, look
for the digestion pattern for the two restriction enzymes.
Compare the size of each fragment with that of the DNA
marker.
8. Restriction digestion patternsof lambda DNA obtained
upon treatment with EcoRIand HindIII are markedly
different which demonstrates the fact that each restriction
enzyme recognizes and cleaves only a specific base
sequenceunique to it. The size of the fragments can be
determined by comparing with that of the DNA marker
ran on the same gel

5. Flow chart: