Download Transform cells and spread plates

pGLO Bacterial
Transformation,
Purification and SDS gel
Timeline Tranformation
Monday1/7 Lecture and discussion
Tuesday 1/8 Transformation Laboratory
Transform cells and spread plates
Wednesday 1/9 (FIRE)
Data Collection and Analysis
Observe transformants and controls
Analyze and interpret results
Inoculation
Picking Colonies and Inoculating Cell Cultures
HW
Extension Activities
Calculate transformation efficiency
Timeline Purification
Thursday 1/10
Purification Phase 1
Bacterial Concentration and Lysis
Tuesday 1/15 Purification Phase 2
Removing Bacterial Debris
Purification Phase 3
Protein Chromatography
Timeline SDS Gel
Thursday 1/17—Prepare Samples, Run gel, stain, destain
(helper stay 7th)
Friday 1/18—View Results, turn in lab
Monday 1/21—post lab quiz, turn in lab
Prep for Transformation
Lab
• 3-7 days prior to lab
o Prepare agar plates, arabinose and ampicillan,(page 13-15)
o Aliquot solutions (page 17)
• 24-36 hr prior (page 16-17)
o Rehydrate E. coli
o Streak starter plates
o Prepare pGLO plasmid
Prep for Lab Purification
• Before Inoculation Lab (Page 6-7)
o Prepare liquid culture media, shaking incubator
• Before Purification Phase 1 (page 8)
o Rehydrate lyszyme
o Set up centrifuge
Prep. For SDS Gel lab
check for supplies
95 water bath
pGLO Bacterial
Transformation
Introduction to Transformation
Transformation Laboratory
Data Collection and Analysis
Calculate Transformation Efficiency
Background information
• Genetic transformation:
Occurs when a cell takes up
and expresses a new piece of
genetic material-DNA
• pGLO plasmid contains GFP
gene and the gene for betalactamase, the ampicillin
resistant gene
• Transformed cells are found on
the LB/amp and LB/amp/ara
plates
Lab Overview
Transformation Tips
• Transform cells and spread plates
o Be careful when handling E. coli: It could cause food poisoning
- Decontaminate work surfaces every day and after any spill
- All contaminated liquid or solid wastes are decontaminated before
disposal
- All persons must wash their hands after handling bacteria and before
exiting the lab
o DO NOT mouth pipet, eat, or drink while in lab
o Wear protective eyewear and gloves
o Use a 10% bleach solution for at least 20 min for sterilization of loops and pipets
• Observe transformants and controls/Analyze and
interpret results
o Observe the color and glow of the bacteria under a UV light
o Fewer colonies of bacteria if amp negatively affected growth
o Equal numbers of colonies on both LB nutrient agar and LB/amp agar if amp
had no effect
o The presence of any colonies on the amp plate would suggest that those
bacteria are resistant to the antibiotic ampicillin
Extension Activity
• Calculate transformation efficiency
• How to determine the extent to which genetic
transformation of E. coli cells was successful
• TIPS:
o Transformation efficiency represents the total number of bacterial cells
that express the green protein, divided by the amount of DNA used in the
experiment
o Transformation efficiency= Total number of colonies growing on agar plate
Amount of DNA spread on the agar plate
Green Fluorescent Protein
Purification
Genetic Transformation Review
Inoculation- Growing a Cell Culture
Purification Phase 1
Purification Phase 2
Purification Phase 3
Background Information
• pGLO plasmid contains GFP
gene and the gene for betalactamase, the ampicillin
resistant gene
• When bacteria was plated onto
LB agar containing arabinose
(LB/amp/ara), GFP was
expressed
• When bacteria was plated onto
LB agar that did not contain
arabinose (LB/amp), the gene
was turned off
• Binding
Wash
Elution
Chromatography Overview
Purification Tips
• Purification Phase 1Bacterial Concentration and
Lysis
o Centrifugation is a technique used to separate molecules on the basis of
size by high speed spinning
o Centrifugation results in a pellet of bacteria found at the bottom of the
tube
o The liqu
• Purification Phase Removing Bacterial Debris
o This final centrifugation step serves to separate the large particles of lysed
bacteria (such as the cell membrane and walls) from the much smaller
proteins, including GFP
o The supernatant will fluoresce bright green upon exposure to UV light
o Carefully remove the supernatant
o id that resides above the pellet is called the supernatant
Purification Tips
• Purification Phase 3 Protein Chromatography
o Bacteria contain thousands of endogenous proteins from which GFP must
be separated
o In chromatography, a cylinder, or column is densely filled with a “bed” of
microscopic beads that will form a matrix through which proteins must pass
before being collected
o GFP “sticks” to the column, allowing it to be separated from the bacterial
contaminants
o Equilibration buffer
Binding buffer
Wash buffer
Elution buffer
GFP Protein on
Polyacrylamide
Gels
Polyacrylamide gels
• Tutorial 23 and 25 from disc.
Background
Information/Tips
• Pages 2-8
• Only 4 stations (1, 3, 5, 7)