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Presentation Restriction Digestion JK
Presentation Restriction Digestion JK
promoter
transcription
termination
Multiple cloning site sequence
(MCS)
Selection
marker
origin of
replication
Basic structure of a Cloning and Expression Plasmid Vector
21-03-2017 BIO122 5
Source: Novagen
Restriction enzymes
5 G 3
3 CTTAA 5
5 AATTC 3
3 G 5
Generates 5 overhang (in the 5 direction)
Blunt ends: subsequent ligation is non- 5 GAA TTC 3
specific 3 CTT AAG 5
Molecular cloning
Type IV REs
Recognizes modified, typically methylated DNA.
How are they named
Restriction enzymes are named by the
organism from which they were first isolated.
EcoRI Eco: E.coli R: I: first
strain enzyme of
that type
isolated
BamHI Bam: Bacillus H: I: first
amyloliquefaciens strain enzyme of
that type
isolated
Sau3A Sau: Staphylococcus 3A:
aureas strain
Restriction Enzyme Digestion
An Enzymatic Unit (u) is defined as the amount of enzyme required
to digest 1 ug of DNA under optimal conditions in 1 hour.
Buffer
Dyes
Agarose
gel
Power Supply
Components of an Electrophoresis System
Electrophoresis Buffer:TAE (Tris-acetate-EDTA) and TBE (Tris-
borate-EDTA) are the most common buffers for duplex DNA.
Establish pH and provide ions to support conductivity.
Loading dye:DNA samples are loaded into a gel AFTER the tank
has been filled with buffer, covering the gel.
Contains a dense substance, such as glycerol,
to allow the sample to "fall" in the well
http://bitesizebio.com/13524/how-to-identify-supercoils-nicks-and-circles-in-plasmid-preps/
Restriction Digestion Analysis
Protocol for restriction digestion
Component Volume
Sample Plasmid (DNA) 3 l
Restriction Enzyme 10X Buffer 2 l
Restriction Enzyme EcoRI 0.5 l
Restriction Enzyme HindIII 0.5 l
Water 14 l
Final volume 20l
Mix gently, close the tube and centrifuge for a few seconds.
Incubate at the 37 deg C (enzymes optimum temperature) for
45 min.
Protocol for Agarose Electrophoresis