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EBBEP Workshop
FP Plasmid Digest Lab
Where the FP came from
There are 6 different FP used for this lab available
through EBBEP
Of the 6 proteins; 3 are from the Aequorea victoria
jellyfish and 3 are from Discosoma striata anemone
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Discosoma striata anemone
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Aiquorea victoria jelly
Structure of FP proteins
All of the proteins used by EBBEP have a similar
structure.They are all monomer barrel shaped
molecules with a central chromatophore
Small changes in the
Amino acids in or directly
Around the
chromatophore cause
changes in color
How were the proteins made?
All proteins were made by Mutagenesis of
the natural occurring proteins found in
types of Cnidarians.
Proteins made from
anemone RFP
Proteins made from
Jellyfish GFP
Tangerine (orange/pink)
Cherry (Dark pink)
Grape (purple)
Emerald (Green)
Venus (yellow)
Blue
Using evolution
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 Since the
proteins come
from two
different “roots”
of evolution you
can see the
inheritance in
the sequences
of the proteins
The FP plasmids
Made from
pRSET vector
FP gene is less
than 730bp
Only difference
in plasmids is
the FP gene
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Restriction enzymes
 Type 2 restriction endonucleases are
used in this lab. (most common)
Cut DNA at a specific sequence.
Enzymes used
 HindIII
 AhdI
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Why are there restriction
enzymes?
Evolved by
bacteria to
protect against
viral infection
Over 3000
known enzymes
Challenges in making the lab
 Needed an enzyme that cut green based
genes different than red based genes.
 That’s easy! Several to choose from
 Finding enzymes that cut the plasmid into
a reasonable number of bands and have
bands of an easily visible size on a gel
 Not so easy
Plasmid maps
Provided are 2 maps (red based and
green based)
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Reading the maps and predicting
size
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 HindIII only cuts
the plasmid once
making 1 linier
piece the full size
of the plasmid
~3623bp
Reading the maps and predicting
size
AhdI cuts 2 times producing
2 fragments
Size of fragment 1
2555-445= 2100bp
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Size of fragment 2 has 2
way to calculate
3623-2110 = 1513bp
Or
3623-2555+445= 1513
Teacher set-up tips
Get labeling tubes early!
DNA, Buffers, Water, loading dye, and
marker/ ladder are directly aliquotted
from stocks supplied
 Can be completed a few days in
advance lf you have a freezer. More if
you have a frosted freezer
Teacher Set-Up tips
Enzymes must be diluted.
 Receive a stock enzyme tube and a dilution
tube for each enzyme.
 Add contents of one tube to the other (pre
measured for you)
 Must be done no earlier than 24 hrs before or
enzymes will not work as well
Teacher set up tips
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 Sterile 1.5 snap cap tubes and filter pipette
tips are ideal.
 Will work without the Sterile but you will
possibly see fuzzier bands in the gel.
 Cap tubes before you give them out to help
decrease the contamination
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Teacher Set-Up tips
Make sure you plan for a long day.
Staining gels with EtBr takes time.
If you don’t have a digital imaging
system then remember you digital
camera.
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Things to stress with students:
 Set up Digests on ICE!
 Concentrations are important (More is not
better)
 Add reagents in Correct order.
 Water first ,Buffer, DNA, Enzymes are always
last.
 Reason for Enzymes being last: Enzymes are
sensitive to conditions outside of their normal
range. Strong buffer solutions can effect the
efficiency of the digest
Lab Procedure
 Each group of 2 students sets up 3 tubes.
 Negative control (no enzyme)
 An undigested plasmid shows as usually 2 or
more bands (nicked/open circle, super super
coiled,multimers
Travels
Faster
Travels
Slower
bs.kaist.ac.kr
Lab procedure
 HindIII digest-Should make 1 linier fragment.
Shows true size of plasmid
 AhdI digest- determines if plasmids are from
red (2 bands) or green (1band) line.
 2 Groups per gel with marker in middle.
 Best to give 1 green and 1 red to each gel.
Lab procedure
Incubate at 37C.
Freeze over night (fridge will work for
24hrs)
Run gel. The bands are far enough
apart you can run it pretty fast… if you
can live with the smile bands on the gel
Keep it clean and don’t digest too long
 Contaminations of DNases can be
devastating.
Lane 8 has little to none
DNase activity
Lane 1 has a large
amount of DNase activity
biosyn.com
Things that can go wrong
Impeded digestion due to incorrect set
up. (too much or too little buffer etc.)
Star digest activity
 Under non-standard reaction
conditions, some restriction enzymes
are capable of cleaving sequences
which are similar but not identical to
their defined recognition sequence.
This altered specificity has been
termed star activity"
Star digestion example
 Examples:
EcoRI is supposed
to only cut GAATTC
but, under extreme
conditions, it might
possibly cut
CAATTC also.
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http://www.fermentas.com/en/support/technical-reference/restriction-enzymes/star-activity
Star digests
Things that can cause Star digests
 Too much glycerol in reaction. (More is not
better)
 Incorrect buffer of buffer concentration
 Extended digest times. Don’t leave them
over night
Expected results
Plasmids made from the Green FP
(jellyfish) digest into single bands for
both HindIII and AhdI
Plasmids made from the Red FP
(anemone) digest into a single band for
HindIII and 2 bands for AhdI
Sample gel from small FP plasmid digest lab
1
2
3
4
5
6
7
8
Multimers
Linier
Nicked circle
2 distinct bands
showing red protein
origins
Red AhdI
Red HindIII
Marker
Undigested red based plasmid
Green AhdI
Green HindIII
Undigested green based plasmid
Supercoiled
Making a standard curve
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Measure to
leading edge of
each band in the
marker/ ladder
Standard Curve
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 Graph of lambda
HindIII marker (base
pair vs. Distance
migrated)
 Using semi log
graph paper
Using graph to find size of
Unknowns
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Standard curves
http://www.phschool.com/science/biolog
y_place/labbench/lab6/standcur.html