1. Classic Procedure for Making Competent Cells

Each student obtain 10 ml of mid-log DH5a cells, and follow the protocol in the Lab Manual (except do NOT flame plastic tubes, pipettes. etc.).

Keep bacterial cells cold during this protocol! This is one of the most important things.

Everyone will have 1 ml of "competent cells" at the end of Part A.

NOTE: All labs will resuspend cells in 1 ml of "magic slime" instead of the 1 ml of CaCl₂.

2. Transformation of E. coli with Recombinant pAMP/pKAN plasmids.

Work in groups of THREE or FOUR (as directed by Instructor). Each group of students will label the following test tubes for addition of competent cells:

• viability control = NO DNA added, add 10 ml of TE
• pAMP = pAMP control
• pKAN = pKAN control
• pLIG #1 = ligation mix from first student
• pLIG#2 = ligation mix from second student
• pLIG #3 = ligation mix from third student
• pLIG #4 = ligation mix from fourth student

Follow the protocol, and add 200 ml of competent cells plus 10 ml of the appropriate DNA sample (or TE buffer) to each tube. Add the 10 ml directly to the competent cells on the bottom of the tube.

Continue through the incubation on ice, heat shock, and recovery steps. We are saving all the remaining competent cells so don't throw them away.

Collect and label your petri plates during incubation periods

PLATE	LB+Amp	LB+Kan	LB+Amp+Kan	LB Only
CODE	(red lines)	(green line)	(red and green)	(black line)
per 4 students	4	4	7	1
per 3 students	3	3	5	1

Label the petri plates properly:

Label on the bottom of the plate, with your lab date, initials of the student pair, and the name of the bacteria and the plasmid. For example, if you transformed the DH5a bacteria with pAMP, then label the petri plate DH5a/pAMP. Bacteria transformed with the ligation mix are labeled DH5a/pLIG. Bacteria without plasmid are labeled DH5a/no DNA.
 

Use the table below as a checklist for spreading the bacterial cells with newly-transformed plasmids onto the petri plates. Check with the instructor if you have any questions!
 

	DH5a Bacteria Transformed With:
Petri Plate:	pAMP control	pKAN control	Ligated DNA pLIG#1	Ligated DNA pLIG#2	Ligated DNA pLIG#3	Ligated DNA pLIG#4	Viability control (no DNA)
LB+Amp	100 ml	100 ml	100 ml				100 ml
LB+Kan	100 ml	100 ml	100 ml				100 ml
LB+Amp+Kan	100 ml	100 ml	100 ml	100 ml	100 ml	100 ml	100 ml
LB Only							100 ml

Add 100 ml 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:

Collect all the competent cells into one ice bucket. SAVE these cells. Put the tubes with bacteria on the rack by the sink and squirt in 30% bleach. Do not bleach the competent cells. Swab down the bench. Wash your hands before leaving the lab.
 

NEXT WEEK THE DAY BEFORE LAB don't forget!!!

Start overnight cultures from your transformation plates.

First 3 students alphabetically- two independent colonies from the LB+Amp plate
Next 3 students alphabetically- two independent colonies from the LB+Kan plate
All remaining students- two independent colonies from the LB+Amp+Kan plate

Pick two independent colonies. Everything you need will be on the lab bench; tubes with LB+Antibiotic (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+Antibiotic, using the sterile yellow plastic loops. Make sure you use the right antibiotic for your cells. 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 37' C in the shaking waterbath. Set shaker at 150-155. Use a plastic test tube rack because the metal ones rust.