1.Classic Procedure for Making Competent Cells
We have competent cells available from earlier in the semester. These have been frozen at -80'C. It is important that the cells be thawed on ice, and kept cold at all times except when specified in the protocol.
 

2. Transformation of E. coli with Recombinant  rDNA/pBLU plasmids.

Everybody will be transforming their own ligation mix (pLIG). Half of the class will also transform control plasmid pBLU, and the other half will do a viability control. These viability cells will be carried through all the steps but NO PLASMID will be added.

Check here if you will transform pBLU_________

Check here if you will do a viability control_________

• If you are transforming pBLU, get four LB/Amp plates from the back of the room (2 red stripes)
• If you are doing a viability control, get three LB/Amp plates from the back of the room and one plain agar plate (no stripes).

Caution - the X-gal is dissolved in an organic solvent. Do not spill! Wash hands immediately if you get any on your skin.

Each students will label the following test tubes for addition of competent cells:

        pLIG = ligation mix (rDNA/pBLU)

        pBLU = pBLU control

OR:

        pLIG = ligation mix (rDNA/pBLU)

        VIA = viability control; NO plasmid

Follow the protocol, and add 200 ml of competent cells plus 10 ml of the appropriate DNA sample to each tube. Keep competent cells cold. Continue through the incubation on ice, heat shock, and recovery steps. The viability control goes through all the steps but no plasmid is added to these cells.

Label the petri plates properly:
Label on the bottom of the plate, with your lab date, your initials, and the name of the plasmid. For example, if the DH5a bacteria has been transformed with 100 ul of pBLU, label the petri plate "pBLU 100". The viability control is plated on the Amp plate and the LB only plate. Label this "VIA".

Make a change in the Protocol at Step #13:
Label eppendorf tubes "L20", "B20" and "VIA". Add 80 ul of sterile LB broth to the eppendorf tubes. Then add 20 ul of the correct competent cells to the labeled eppendorf tube. Gently mix with the pipettor, then add the entire contents to the petri plate. Spread cells as usual. Remember that the viability control is plated on the LB only plate.

Add 100 ul of cell suspension to each plate and spread until ALL the liquid has been absorbed. Keep spreading until the plate is dry. This is an important step to get isolated colonies.

Be very careful with the alcohol and the open flame!
 

Cleanup:
Put the tubes with bacteria on the rack by the sink and squirt in 30% bleach. Swab down the bench. Wash your hands before leaving the lab.

NEXT WEEK THE DAY BEFORE LAB donít forget!!!

Start overnight cultures from your transformation plates.

Pick two independent WHITE colonies from the DH5a/pLIG plates. Everything you need will be on the lab bench; tubes with LB+Amp (extra tubes are in the refrigerator), your transformation plates, and sterile plastic loops.

Use sterile technique and pick two separate colonies Each colony is inoculated into a separate tube of LB+Amp, using the sterile yellow plastic loops. Put used loops in glass jar marked "loop disposal".

Use tape to label tubes. Put tape near the top of the tube so the cap will cover the tape (this prevents it from coming loose in the waterbath). Push caps down firmly with a twisting motion. The tubes are incubated overnight at 37íC in the shaking waterbath. Set shaker at 150-155. Use a plastic test tube rack because the metal ones rust.