LAB report 12 HOURS( 2 PAGES)

Biol 390-Lab 7 Select transformants and isolate plasmids

Objective

· Select transformed cells.

· Freeze cells for future labs.

· Isolate plasmids from transformed cells

Background

p-GLO plasmid

pGLO contains several DNA sequences that enable replication of the plasmid DNA and expression of the fluorescent trait (phenotype) in bacteria following transformation. The essential sequences include the following:

GFP — the jellyfish gene that codes for the production of green fluorescent protein

bla  — a gene that encodes the enzyme beta-lactamase, which breaks down the antibiotic ampicillin. Bacteria containing the bla gene can be selected by placing ampicillin in the growth medium.

ori — the origin of pGLO plasmid DNA replication

araC  — a gene that encodes the regulatory protein that binds to the pBAD promoter. Only when arabinose binds to the AraC protein is the production of GFP switched on.

pBAD promoter — a specific DNA sequence upstream from the GFP gene, which binds araC-arabinose and promotes RNA polymerase binding and transcription of the GFP gene.

Multiple cloning site — a region containing restriction sites (NdeI, HindIII, EcoRI, etc.), sequences that permit the insertion or deletion of the gene of interest

Materials

For day before

· Three tubes of LB broth / group

· Ampicillin solution (10mg/ml)

· Sterile plastic loops

Day of the lab

· Sterile tips

· Bleach spray bottles

· Nitrile gloves

· 20% glycerol sterile

· Sterile tubes for freezing cultures

· Midisci high speed plasmid mini kit

· Add RNase A to PD1 buffer and store at 4 oC (prepare day before)

· Check for precipitates in PD2 buffer – if present warm in 37oC bath

· Add 100 ml of absolute ethanol to wash buffer

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Procedure

Day before

· Add ampicillin to LB tubes containing 5 ml of LB broth.

· How much ampicillin do you need to add to make the final concentration 100ug/ml

· Label LB tubes with your name an number for each colony.

· Inoculate three separate colonies into three different tubes of LB. Pick three colonies from the LB amp/ara using sterile a sterile loop for each transfer.

· Incubate tubes at 37oC

Day of lab

· Observe plates from your experiment. Where possible count the number of colonies. Which colonies glow?

Freeze cells

· Label 3 sterile Eppendorf tubes with your name and your colony number.

· Aseptically transfer 0.5 ml of your overnight culture to a sterile Eppendorf.

· Using a new pipette add 500ul of 20% glycerol

· Freeze at -80oC

High-Speed Plasmid Mini Kit Protocol -Midisci

· Add provided RNase A to the PD1 Buffer and store at 4oC. (the day before)

· If precipitates have formed in the PD2 Buffer, warm the buffer in a 37oC water bath, followed by gentle shaking to dissolve.

· Add absolute ethanol to the Wash Buffer prior to initial use (see the bottle label for volume).

· Additional requirements: microcentrifuge tubes.

Step1 Harvesting

· Transfer 1.5 ml of cultured bacterial cells to a microcentrifuge tube. Microcentrifuge for 1 minute and discard the supernatant.

Step 2 Re-suspension

· Add 200 μl of PD1 Buffer (RNase A added) to the tube and resuspend the cell pellet by vortex or pipetting.

Step 3 Lysis

· Add 200 μl of PD2 Buffer and mix gently by inverting the tube 10 times. Do not vortex to avoid shearing the genomic DNA. Let stand at room temperature for 2 minutes or until the lysate is homologous.

Step 4 Neutralization

· Add 300 μl of PD3 Buffer and mix immediately by inverting the tube 10 times. Do not vortex .

· Microcentrifuge for 3 minutes.

Step 5 DNA Binding

· Place a PD Column in a 2 ml Collection Tube.

· Add the supernatant from Step 4 to the PD Column and microcentrifuge for 30 seconds.

· Discard the flow-through and place the PD Column back in the 2 ml Collection Tube.

Step 6 Wash

· Add 400 μl of W1 Buffer into the PD Column.

· Microcentrifuge for 30 seconds.

· Discard the flow-through and place the PD Column back in the 2 ml Collection Tube. Add 600 μl of Wash Buffer (ethanol added) into the PD Column. Microcentrifuge for 30 seconds.

· Discard the flow through and place the PD Column back in the 2 ml Collection Tube. Microcentrifuge again for 3 minutes to dry the column matrix.

Step 7 DNA Elution

· Transfer the dried PD Column to a new microcentrifuge tube.

· Add 50 μl of Elution Buffer or TE into the center of the column matrix.

· Let stand for 2 minutes or until the Elution Buffer or TE is absorbed by the matrix

· Microcentrifuge for 2 minutes to elute the DNA.

· Store at -20oC

Data Analysis

· Explain that in the +pGlo experiments why the colonies that grew on the LB/amp/ara fluoresced, while those that grew on LB/amp did not

· Why do colonies of the –Pglo not grow on the LB/amp plate

References

· MIDIsci

· http://shop.midsci.com/protocals/IB47100Protocol.pdf

· Biorad

· http://www.bio-rad.com/en-us/applications-technologies/pglo-plasmid-map-resources

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