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Bacterial Genetics: Transformation
Collect 1 LB, 2 LB/Amp, and 1 LB/Amp/Ara plate from the side bench. Also collect 2, sterile microcentrifuge tubes.
- Label the plates as follows
LB plate: "- DNA"
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LB/Amp plate: "- DNA"
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LB/Amp plate: "+ DNA"
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LB/Amp/Ara plate: "+ DNA" 
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- Label one microcentrifuge tube "- DNA" and the other "+ DNA".
Place the microcentrifuge tubes on ice.
- Using a P-20 pipetman, carefully pipette 10 microleter of pGLO plasmid into the microcentrifuge tube labeled "+DNA".
- Make sure you see a tiny drop of fluid in the bottom of the tube before going on.
Collect a tube of competent Escherichia coli from the TA or instructor. These cells will be frozen. It is important to let the cells thaw on ice and then proceed with step 4 as soon as they thaw.
- Using a P-200 pipetman, add 200 microleter of competent E. coli to the "- DNA" tube. Place the tube back on ice.
- Using the P-200, add 200 microleter of competent E. coli to the "+ DNA" tube and place it back on ice.
- Tap the "+ DNA" tube gently to mix.
- Incubate both tubes on ice for 20 minutes.
- Heat shock the cells by placing both tubes in a 42° C water bath for 30 seconds. Place both tubes back on ice.
- Using a P-1000, add 500 microleter of LB/20mM glucose media to each tube.
- Allow the cells in both tubes to recover from the heat shock by incubating them in a 37° C water bath for 30 minutes.
- Using a P-200 pipetman...
-Pipette 100 microleter from "- DNA" tube onto the LB plate.
-Pipette 100 microleter from "- DNA" tube onto one LB/Amp plate.
-Pipette 100 microleter from "+ DNA" tube onto the other LB/Amp plate.
-Pipette the remaining volume from "+ DNA" tube onto the LB/Amp/Ara plate.
- Using a flamed, sterile loop, spread the volume evenly over the agar surface by running the loop side to side across the plate. Rotate the plate 90° and repeat this pattern. Do this several times to ensure that the entire surface of the plate is covered.
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Be sure to flame the loop before and after spreading each plate.
- Allow the plates to dry for 5 – 10 minutes and place all four plates upside down in the 37°C incubator for 24 hours.
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