EBBEP Workshop
FP Plasmid Digest Lab
Where the FP came from
There are 6 different FP used for this lab available through EBBEP
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Of the 6 proteins; 3 are from the Aequorea victoria jellyfish and 3 are from Discosoma striata anemone
Discosoma striata anemone 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 directlyAround 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 Jellyfish GFPEmerald (Green)Venus (yellow)Blue
Proteins made from anemone RFPTangerine (orange/pink)Cherry (Dark pink)Grape (purple)
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
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Made from pRSET vector
FP gene is less than 730bp
Only difference in plasmids is the FP gene
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 labNeeded 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
HindIII only cuts the plasmid once making 1 linier piece the full size of the plasmid ~3623bp
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Reading the maps and predicting size
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AhdI cuts 2 times producing 2 fragments
Size of fragment 12555-445= 2100bp
Size of fragment 2 has 2 way to calculate
3623-2110 = 1513bp Or3623-2555+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
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. QuickTime™ and a
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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
TravelsFaster
TravelsSlower
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 longContaminations of DNases can be devastating. Lane 8 has little to none
DNase activityLane 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 activityUnder 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 digestsToo 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
Nicked circle
1 2 3 4 5 6 7 8
Undigested green
base
d plasm
id
Undigested red based pla
smid
Gre
en Hind
III
Red H
indIII
Gre
en AhdI
Red A
hdI
Marker
Supercoiled
Multimers
2 distinct bands showing red protein origins
Sample gel from small FP plasmid digest lab
Lin
ier
Making a standard curve
Measure to leading edge of each band in the marker/ ladder
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Standard Curve
Graph of lambda HindIII marker (base pair vs. Distance migrated)
Using semi log graph paper
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Using graph to find size of Unknowns
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Standard curves
http://www.phschool.com/science/biology_place/labbench/lab6/standcur.html