Restriction Digest
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2
Restriction Enzymes/Endonucleases
• Restriction Enzyme/Endonuclease - recognizes
short DNA sequences and cleaves double stranded
DNA at a particular sequence.
– The recognition sequences are generally 4 to 6 nucleotides
in length.
– Called an ‘endonuclease’ because it cleaves within a
nucleotide sequence.
• Found naturally in bacteria.
– Used by bacteria to degrade foreign DNA (bacteriophage).
– Bacteria protect their own chromosomal DNA by methylating
the nucleotides within the recognition sequence.
Restriction Enzymes/Endonucleases
• Found naturally in bacteria.
– Used by bacteria to degrade foreign DNA (bacteriophage).
– Bacteria protect their own chromosomal DNA by methylating
the nucleotides within the recognition sequence.
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Restriction Enzymes/Endonucleases
• EcoRI from Escherichia coli
• BamHI from Bacillus amyloliqueraciens
• PvuI and PvuII are different enzymes
from same strain.
• Originally purified by individual labs,
Nathans, Smith
• Now supplied by companies - GE, NEB,
Promega, BRL
Restriction Enzymes/Endonucleases
• “flush” or “blunt” ends
– DraI cleavage generates blunt ends:
5´ T-T-T≠A-A-A 3´
3´ A-A-A≠T-T-T 5´
• “sticky ends.”
– KpnI cleavage generates cohesive 3´ overhanging
ends:
5´ G-G-T-A-C≠C 3´
3´ C≠C-A-T-G-G 5´
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Restriction Enzymes/Endonucleases
Restriction Enzymes/Endonucleases
• Degenerate (Recognizes multiple sequences):
• AvaII GGWCC:
GGTCC, GGACC
• AvaI CPyCGPuG
Py stands for pyrimidine- T or C
Pu stands for purine - A or G
CTCGAG
CTCGGG
CCCGAG
CCCGGG
• DdeI CTNAG: CTAAG, CTGAG, CTCAG, CTTAG
• BbsI cleaves
GAAGACNN
CTTCTGNNNNNN
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Restriction Enzymes/Endonucleases
Restriction Enzymes/Endonucleases
• It is possible that two or more restriction endonucleases
recognizing different sequences may generate compatible
ends.
– BgIII and BamHI recognize two different sequences but
produce compatible ends.
BamHI
BglII
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Restriction Enzymes/Endonucleases
• Mg+2 or Ca+2 is requirement for enzyme activity.
• A buffer is necessary in order to maintain the optimal pH for
the enzyme function.
• When more than one restriction endonuclease is used in a
reaction a buffer compatible with both enzymes must be
used.
Restriction Enzymes/Endonucleases
• How much enzyme to use
– Unit = Amount of enzyme required to digest 1µg of
DNA in 1hr under optimal conditions
– Generally 5-10 Units are used per digestion
• How much DNA to use
– .1-5 µg
• Use the correct buffer
• Incubation time (1-2 hrs)
• Incubation temperature (generally 37°C)
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Restriction Enzymes/Endonucleases
• As a general rule of thumb the concentration of DNA
obtained from the mini prep is approximately .1µg/µl.
• We will digest 10µl of DNA from the mini prep. 10µl of
mini prep DNA is 1 µg of DNA.
• A Unit of enzyme is the amount of enzyme required to
digest 1µg of DNA in 1hr under optimal conditions and
1µl of enzyme is about 10 Units.
• Will this be enough to digest our concentration of DNA?
• What is the minimum amount of enzyme required for
each sample for our concentration of DNA? How many
Units of enzyme is this?
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• Restriction enzymes can be used to determine the
size of the Wolffia cDNA insert.
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Important!
• Enzymes should be stored at -20°C.
• While in use, enzymes should be
carefully maintained on ice.
• Be careful to avoid contaminating
enzyme solutions.
• Add the enzyme last just in case you
make any mistakes with the other
reagents!
Today’s Experiment
• Digest the purified plasmid DNA in order to determine the
size of the insert
• Use the restriction endonuclease PvuII to digest the
plasmid
• Complete a digest for each mini prep.
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Today’s Experiment
• Master Mix
– Label a microfuge tube as
“5-Rxns. Digest Mix.” In
this tube prepare a
reaction digestion mixture.
– Notice that there will be
more mix than you will
need (for 5 rather than 4
reactions). This is done to
allow for pipetting error so
you don’t run out of mix.
Today’s Experiment
•
•
Mix the ingredients of the
“5-Rxns Digest Mix” by
gently pipetting the mix up
and down a few times
until mixed. (Do Not
Vortex!)
Aliquot 10 ul of the “5Rxns. Digest Mix” to each
of four fresh,
appropriately labeled
microcentrifuge tubes,
then add 10 ul of each
plasmid DNA to obtain a
final reaction volume of
20 ul.
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Today’s Experiment
• Gently mix each digest by tapping the tube.
• Place your digest tubes in a sample rack and incubate the rack
of samples in the 37ºC water bath for 5 to 10 minutes.
• When your digests are done incubating, store your samples in
your –20ºC freezer box until you are ready to load them onto an
agarose gel.
Today’s Experiment
• REMEMBER: KEEP THE RESTRICTION
ENZYME ON ICE AT ALL TIMES!
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Theoretical PvuII digests of plasmids
p. 2B-10
Gel Electrophoresis
• Uncut DNA is used as a control.
• Three bands appear in the uncut lane.
• Open circle/Nicked DNA - Nicked plasmid DNA runs about 2x
slower than linear DNA
• Linear DNA - Some of the plasmid DNA gets cut open during
the mini prep and runs to its true size.
• Supercoil - Uncut plasmid DNA runs faster than linear or
nicked DNA
4-4
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A real Digest gel of clones from the Duckweed cDNA library
p. 2B-11
1.Did the gel run OK?
A) Yes
B) No
C) Can’t tell
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2.Is there DNA in your minipreps?
Are there bands in the uncut sample?
A) Yes
B) No
C) Can’t tell
p. 2B-11
3.Is there the same level of DNA in
your minipreps?
A) Yes
B) No
C) Can’t tell
p. 2B-11
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4. Did your DNA cut?
Does your digested sample look different than your uncut?
A) Yes
B) No
C) Can’t tell
p. 2-18
5. Does your digest look complete?
Are the intensities of the bands proportional to the size?
p. 2B-12
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Bands Generated from Partial Digests
p. 2-19
6. Is there a PvuII site in the insert?
Are there multiple bands?
p. 2B-12
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6. Is there a PvuII site in the insert?
Are there multiple bands?
p. 2B-13
7. What is the size of the insert?
2.9 Kb
700 bp - 700 bp = 0 bp
p. 2B-13
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8. Do the results from the digest and the
PCR agree?
700 bp - 700 bp = 0 bp
200 bp - 200 bp = 0 bp
p. 2B-14
8. Do the results from the 20JM1.10
digest and the PCR agree?
1100 bp - 700
bp = 400 bp
600 bp - 200 bp = 400 bp
p. 2B-14
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8. Do the results from the 20JM2.10
digest and the PCR agree?
2200 bp - 700 bp
= 1500 bp
1700 bp - 200 bp = 1500 bp
p. 2B-14
8. How do we figure out the insert size
from the 20JM4.10 digest?
2900 bp
1200 bp
400 bp
1200 bp + 400 bp700 bp = 900 bp
p. 2B-14
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8. Can we map the position of the PvuII
site?
A)
2900 bp
1200 bp B)
400 bp
p. 2B-14
8. Do the results from the 20JM4.10
digest and the PCR agree?
1100 bp - 200 bp = 900 bp
1200 bp +400 bp700 bp = 900 bp
p. 2B-14
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Home Work!! Fill
in the rest of the
table for Chapter
2B Problem Set
Protocol
• Wear gloves! There is
ethidium bromide in the
buffer!
• Make 70ml of a 1%
agarose in 1x TBE. Use
an Erlenmeyer flask for
this solution.
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Protocol
• Use a Kim wipe or a piece of
paper towel as a stopper for
the flask and heat the solution
in the microwave for ~2
minutes.
Protocol
• Make sure that the agarose
completely dissolves.
• Allow to cool to ~60ºC. (This
should take a couple of
minutes. Estimate by touch)
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Protocol
• Pour the solution onto the
casting tray.
• Add the comb to the liquid
agarose.
• Allow to set. (~30 minutes)
Protocol
• Carefully remove the
comb from the gel
• Place the gel into
the electrophoresis
chamber in the
proper orientation so
that the negatively
charged DNA runs
toward the anode
(+)
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Protocol
• Add enough 1x TBE
to cover the gel
Protocol
• Obtain your restriction
digests from the
freezer and add 2 µl
of 10X Gel Loading
Dye Mix to each of
your four restriction
digest tubes.
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Protocol
• Obtain uncut mini prep DNA from the
freezer
Protocol
• Arrange the
samples in the
order you will load
the gel.
• Record this
information in your
lab notebook.
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Protocol
• Load the DNA samples
(all are approx. 20 µl)
into wells using a P-20
pipetman set at 20 µl.
In addition, load one
lane of 10 µl of premade DNA size
standards known as
1Kb Plus DNA Ladder.
Protocol
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Protocol
• Attach the lid to
the gel box.
• Plug the leads
into the power
supply and set it
to 100V.
• Turn on the
power supply
and press the
run button.
Protocol
• After you have
turned on the gel,
make sure that
bubbles are coming
from the wire
electrodes,
indicating that there
is current in the gel.
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Protocol
• Turn off the power supply after the
tracking dye runs about 2/3 of the way
down the gel.
Protocol
• Remove the gel
from the chamber
(Be sure to keep
track of the gel’s
orientation).
• Place the gel on
the UV light box.
• Take a picture.
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Protocol
• Tape the picture into
your lab notebook.
• Label the gel and
analyze the bands in
each lane.
• Record your results.
Protocol
• Download the
RDG PowerPoint
template from the
website.
• Crop and mark
up your gel
• Save your gel
using the proper
naming format
• Upload your
marked up gel to
the Sakai drop
box.
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