Download Plasmid

Plasmid DNA restriction
and
agarose gel electrophoresis
Laboratory work Nr.3
Plasmid
A plasmid is a small circular, doublestranded DNA molecule that is
 physically separated from a chromosomal DNA
 can replicate independently
 found mostly in bacterial cells
 carry genes that benefit the survival of the
organism (antibiotic resistance).
Vector
Artificial plasmids are widely used
as vectors in molecular cloning,
in order to drive the replication
of recombinant DNA sequences within
host organisms.
Their size can range from 1000 to 10000 bp.
Plasmid can be used for gene transfer into human cells so
that it may express the protein that is lacking in the cells.
Restriction enzymes
Several thousand of restriction enzymes have been
isolated from bacteria, where they appear to serve a
host-defense role.
The idea is that foreign DNA, for example from an infecting virus,
will be chopped up and inactivated ("restricted") within the
bacterium by the restriction enzyme.
The substrates for restriction enzymes are more-or-less specific
sequences of double-stranded DNA called recognition
sequences.
Restriction enzyme digestion is a commonly used technique for
molecular cloning, It is also used to quickly check the identity of
a plasmid by diagnostic digest.
pOX2R-GFP2-N2
pUC ori
pCMV
HindIII (653)
TK poly (A) signal
Zeocin
resistance
gene
hOX2R
phOX2R-GFP2-N2
5641 bp
ZeoR
human
Orexin
Receptor 2
gene insert
BamHI (1991)
PSV40/Pamp
f1 origin
GFP2 gene
SV40 early poly (A) signal
Recombinant human orexin receptor 2 has been cloned into pGFP2
vector by using BamHI and HindIII restriction sites.
pUC ori
pCMV
HindIII (653)
TK poly (A) signal
Zeocin
resistance
gene
hOX2R
phOX2R-GFP2-N2
5641 bp
ZeoR
human
Orexin
Receptor 2
gene
BamHI (1991)
PSV40/Pamp
f1 origin
GFP2 gene
SV40 early poly (A) signal
Human embryonic kidney (HEK 293) cells expressing Orexin 2 receptor
in cell membrane.
The aim of laboratory work is to confirm the
insertion of recombinant human orexin 2 receptor
sequence into pGFP2 plasmid by using restriction
analysis.
pUC ori
pCMV
HindIII (653)
TK poly (A) signal
hOX2R
phOX2R-GFP2-N2
5641 bp
1339 bp
ZeoR
BamHI (1991)
PSV40/Pamp
f1 origin
SV40 early poly (A) signal
Recombinant
Human
OX2R
GFP2 gene
Expected size of
OX2R fragment is 1339 bp.
Protocol
Restriction digest of the plasmid pOX2R-GFP2
1. You will do a double digest – DNA plasmid digesting with 2
enzymes at the same time.
You will need to determine the best buffer that works for both of
your enzymes- BamHI and HindIII, by reading the instructions
for your enzymes (in Fermentas catalog).
Protocol
Restriction digest of the plasmid pOX2R-GFP2
2. In 0.5ml tube combine the following reagents:
14.2 µl
2 µl
2 µl
1 µl
0.8 µl
H2 O
10x Tango restriction buffer
pOX2R-GFP2 plasmid (1.6 µg/µl)
Hind III enzyme (10U/µl)
BamH I enzyme (10U/µl)
The total reaction volume would be 20 µl.
3. Mix gently by vortex and spin down all the pellets.
4. Incubate the tube at +370C for 40 min.
5. To visualize the results of your digest, conduct agarose gel
electrophoresis.
Gel electroforesis of restricted plasmid and
PCR products (from Lab.work Nr2)
100bp marker
Electrophoresis uses an electrical field to move the negatively
charged DNA toward a positive electrode through an agarose gel matrix.
The gel matrix allows shorter DNA fragments to migrate more
quickly than larger ones.
1.
2.
3.
4.
5. -cont.
Thus, you can
accurately determine
the length of a DNA
segment by running it
on an
agarose gel alongside a
DNA ladder (a
collection of DNA
fragments of known
lengths).
Gel electroforesis of restricted plasmid and
PCR products (from Lab.work Nr2)
DNA visualization in agarose gel is done by EtBr (bind to DNA).
When exposed to UV light, it will fluoresce with an orange
colour, intensifying after binding to DNA.
EtBr is a known mutagen. Wear a lab coat
and gloves when working with EtBr!!!!!!
Protocol
Pouring a 1% agarose gel
1. Measure out 1 g of agarose and add 100 ml of 1x TAE buffer.
2. Microwave for 40 sec, stop and swirl, and then continue
towards a boil (until agarose is completely dissolved). Be careful
stirring, eruptive boiling can occur!
3. Let agarose solution cool down for 3 min.
4. Add 7 µl ethidium bromide (EtBr) from lab stock solution to
agarose solution.
5.Pour the agarose into a gel tray with the well comb in place.
Pour slowly to avoid bubbles which will disrupt the agarose gel.
6.Let the gel sit for 20 min, until it has completely solidified.
Protocol
Loading samples and running an agarose gel.
Loading buffer serves two purposes:
1) it provides a visible dye that helps with gel loading and will also
allow you to gauge how far the gel has run while you are
running your gel; and
2) 2) it contains a high % glycerol, so after adding it your sample is
heavier than water and will settle to the bottom of the gel well,
instead of diffusing in the buffer.
Loading samples and running an agarose gel.
Analysing your PCR and restriction DNA fragments
Using the 100 bp DNA ladder in the first line as a
guide, determine
 the size of your GCK4 and HNF1A gene fragments
from PCR, and
 the size of recombinant OX2R from plasmid
restriction.