due 24 hours
jacckkyy
exp2_day2.pdf
Welcome
Experiment II Day 2
September 15, 2016
Lab Reports are due on Sept. 20!
Conjugation
Transduction
Conjugation:
• First, we need to get the cultures you inoculated Tuesday growing again.
• Dilute cultures1:5 dilutions of each in a separate tube, (for example, 1 ml of culture
in 4 mls sterile LB).
Conjugation: Use diluted cultures to set up the following tubes:
Label three 13x100 mm tubes (Do not use mirofuge tubes at this point). • Tube 1: Mix 500µl E. coli Hfr KL16 and 1ml of rif mutant.
• Tube 2: 1ml rif mutant.
• Tube 3: 500µl of KL16.
• Incubate without shaking @ 37°C for 90 min.
• While waiting, prepare plates with 50µl of each amino acid:
Transduction: • Streak your rif mutant (from original
culture, not from diluted culture) on a fresh LBrif plate incubated at 37°C.
• Streak your lysogen on a fresh LB Cm plate incubated at 30°C.
TRYPTOPHAN
ARGININE ARGININE
HISTIDINE
ARGININE
TRYPTOPHAN TRYPTOPHAN
HISTIDINE
TRYPTOPHAN
ARGININE
Donor Mixture Recipient Mixture KL16: his+trp+arg+strSrifS HR171: his-trp-arg-strRrifR
Conjugation: • Transfer cultures from incubation tubes to
microcentrifuge tubes: – 0.5 ml E. coli KL16 – 1.0 ml rif mutant – 1.5 ml mixture
• Centrifuge 60 sec (balance!) to pellet cells. • Pour off supernatant (Carefully). • Resuspend in 350µl saline. • Centrifuge AGAIN for 60 sec and resuspend in
350µl saline AGAIN.
• Spread 100µl of each culture to designated plates:
• Be sure to rinse your spreader with water when changing amino acids.
• Incubate plates at 37°C.
TRYPTOPHAN
ARGININE ARGININE
HISTIDINE
ARGININE
TRYPTOPHAN TRYPTOPHAN
HISTIDINE
TRYPTOPHAN
ARGININE
Donor Mixture Recipient Mixture KL16: his+trp+arg+strSrifS HR171: his-trp-arg-strRrifR
Clean-Up • Microfuge tubes go in tall orange containers on benches.
• Glass tubes go in the back under “discard tubes here” sign: – Separate lrg. and sm.
• Please throw away any paper towel, trash, parafilm wrappers, etc.
• BE SURE YOUR PLATES FOR NEXT WEEK ARE AT THE CORRECT TEMPS!
