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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. Aiquorea victoria jelly. Discosoma striata anemone.

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ebbep workshop

EBBEP Workshop

FP Plasmid Digest Lab

where the fp came from
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

Aiquorea victoria jelly

Discosoma striata anemone

structure of fp proteins
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.How were the proteins made?

Proteins made from

anemone RFP

Tangerine (orange/pink)

Cherry (Dark pink)

Grape (purple)

Proteins made from

Jellyfish GFP

Emerald (Green)

Venus (yellow)

Blue

using evolution
Using evolution
  • Since the proteins come from two different “roots” of evolution you can see the inheritance in the sequences of the proteins
the fp plasmids
The FP plasmids
  • Made from pRSET vector
  • FP gene is less than 730bp
  • Only difference in plasmids is the FP gene
restriction enzymes
Restriction enzymes
  • Type 2 restriction endonucleases are used in this lab. (most common)
  • Cut DNA at a specific sequence.
  • Enzymes used
    • HindIII
    • AhdI
challenges in making the lab
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
Plasmid maps
  • Provided are 2 maps (red based and green based)
reading the maps and predicting size
Reading the maps and predicting size
  • HindIII only cuts the plasmid once making 1 linier piece the full size of the plasmid ~3623bp
reading the maps and predicting size1
Reading the maps and predicting size

AhdI cuts 2 times producing 2 fragments

Size of fragment 1

2555-445= 2100bp

Size of fragment 2 has 2 way to calculate

3623-2110 = 1513bp

Or

3623-2555+445= 1513

teacher set up tips
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 tips1
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 tips2
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
teacher set up tips3
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.
things to stress with students
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
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 procedure1
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 procedure2
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
slide21
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
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
Star digestion example
  • Examples:
    • EcoRI is supposed to only cut GAATTC but, under extreme conditions, it might possibly cut CAATTC also.

http://www.fermentas.com/en/support/technical-reference/restriction-enzymes/star-activity

star digests
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
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
slide26

Sample gel from small FP plasmid digest lab

1 2 3 4 5 6 7 8

Multimers

Linier

Nicked circle

Supercoiled

2 distinct bands showing red protein origins

Red HindIII

Green AhdI

Red AhdI

Green HindIII

Undigested green based plasmid

Undigested red based plasmid

Marker

making a standard curve
Making a standard curve
  • Measure to leading edge of each band in the marker/ ladder
standard curve
Standard Curve
  • Graph of lambda HindIII marker (base pair vs. Distance migrated)
  • Using semi log graph paper
standard curves
Standard curves
  • http://www.phschool.com/science/biology_place/labbench/lab6/standcur.html