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Lab BT2
pGLO TRANSFORMATION of E. coli
You will genetically engineer the gene for GFP into bacteria,
changing the genotype and phenotype of transformed cells.
Designed for two 90 minute lab periods. Adapted from BioRad
Explorer‰ series for use with micropipets.
PROCEDURE
DAY 1:
Label one closed, STERILE reaction tube “+DNA” and a secondreaction tube “–DNA.” Label both tubes with your team initials.
One at a time, open each reaction tube and transfer 200 µL ofsterile Transformation Solution (50 mM CaCl ) into each tube. [This Close the tubes and place them on ice. (Be sure tubes are uprightand do not tip over if there is too much water in the ice cups.) Use a sterile loop to pick up one single colony of bacteria from astarter plate. Open up the “+DNA” tube and immerse the loop intothe Transformation Solution at the bottom of the tube. Spin the loopbetween your index finger and thumb until the entire colony comesoff the loop. Close the top and “finger flick” or “rack” the tube todisperse the bacteria until you can see no floating clumps. Place thetube back in ice.
With a new loop, repeat step 4 with a colony for the “–DNA” tube.
Remember to keep cells/tubes on ice between manipulations.
Using sterile technique, add 10 µL of pGLO DNA to the cellsuspension in the “+DNA” tube. Close the tube and gently fingerflick it to mix the contents. Place the tube back on ice.
To the “–DNA” tube add 10 µL of sterile water. Close the tube,finger flick the tube and place on ice. [Why is water added to thistube?] Let the tubes sit in ice for 10 minutes. Do not shake until step 11.
Take your tubes IN THE ICE CONTAINER to the 42 °C water bath.
Put BOTH the (+) and (–) tubes into the rack in the water bath forexactly 50 seconds. After the 50 seconds, immediately place bothtubes back on ice. (For the best transformation results, the changefrom the ice to 42°C and then back to the ice must be rapid.)***Keep tubes on ice for at least 2 minutes.*** Digital File Reference Number: LBT20902block.doc Remove tubes from ice and place in a rack at roomtemperature. Open the +DNA tube and add 200 µL of LBbroth to the tube. Close the tube and gently finger flick tomix contents.
Repeat Step 11 for the “–DNA” tube.
Place both tubes in a rack at room temperature for 15minutes.
Obtain 3 petri plates per team:1 LB agar plate1 LB agar + ampicillin plate1 LB agar + ampicillin + arabinose plate Label each plate on the BOTTOM ALONG THE OUTSIDE EDGE, with your team initials, your class period,“+DNA” and the type of plate. PRINT SMALL. You may abbreviate ampicillin as AMP and arabinose asARA. [Why do you label the plates on the bottom? Why print small and along the edges?] Finger flick your “+DNA” tube to mix the cells. Work with oneplate at a time. Using a new sterile tip for each plate, pipet50 µL of the “+DNA” mix onto the LB plate.
Just before you are ready to use it, open your packet ofsterile plastic loops at the end with the point (opposite theloop). Remove loop, holding it on the end. Do not let ittouch anything. Do NOT heat it! Spread the 50 µL of E. coli evenly around the surface of theagar by quickly and LIGHTLY moving the spreader back andforth across the plate surface until all the liquid is absorbed.
Try to completely cover the surface of the agar.
Repeat Steps 16, 17 and 18 for the remaining two plates.
Stack your plates and tape them together. Place the stackupside down in the 37 °C incubator until the next day (or 32°C over the weekend).
CONTROLS ***Two teams will be selected to plate their “–DNA” controlsamples on behalf of the rest of the class. These are theCLASS NEGATIVE CONTROLS. [Why are these controlsneeded?] Obtain and label 3 plates as in Step 14, but indicate“–DNA” along with the other info. Then follow Steps 16 - 18using the “–DNA” tube of cells. Be prepared to share yourresults with the class.
Digital File Reference Number: LBT20902block.doc CLEAN UP. Leave your station neater than you found it.
Obtain your team plates. Observe your set of “+DNA”plates under room light and with UV light. Recordnumbers of colonies and color of colonies.
Observe the two class sets of CONTROL “–DNA”plates, done by one or two of your class teams.
Design a table to organize ALL of the following data: Number of colonies on “+DNA” plates for LB, LB/AMP and LB/AMP/ARA plates.
Number of colonies on “–DNA” plates for LB, LB/AMP and LB/AMP/ARA plates.
What two new traits do your transformed bacteria have? Estimate how many bacteria were in the 50 mL of bacteria that you spread on each plate. Explain yourlogic.
What is the role of arabinose in the plates? List and briefly explain three current uses for GFP (green fluorescent protein) in research or appliedscience.
Give an example of another application of genetic engineering.
Digital File Reference Number: LBT20902block.doc

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