Week 2

Conjugation procedure:
Prep - Add 5ml of LB broth to a test tube followed by KG4. Repeat with KG15 and KG18 (separate tubes so as to avoid recombinants) as well as 6µl kanamycin in both the KG15 and KG18.
KG4 = WT
KG15 = Helper
KG18 =Transposon

Step 1: In order to get bacteria to the exponential phase, 5ml of LB broth is added to a test tube followed by 100µl of suspended bacteria. This is done for all three.
Step 2: Allow bacteria to grow for 3 hours.
Step 3: Add to a 1.5ml tube according to the chart

F, G, and H provide controls for the experiment to assure that the growth we see in the LB AmpKan plates represents recombinants and not simply an error in experimetal design.
Step 4: Centrifuge each tube for 60 seconds @13000rpm then discard the majority of the liquid. Leave just enough to resuspend using the inhale/exhale technique.
Step 5: Spot onto an LB plate to allow for growth overnight.
Wait Overnight
Step 6: Scrape bacteria and resuspend into 1ml of LB Broth
Step 7: Add 200µl each onto 5 LB AmpKan plates (to select against WT)
Allow to grow
Step 8: Replica plate A-E onto Lept, LB Mn, and Lept Kan

6-17
Checked plates A through H for oxidation and noticed none on the LBMn or LeptKan, but did notice oxidation on every Lept plate. I scraped the plates with a sterile toothpick then re-plated onto both a lept and a LBKan plate. The purpose of this is to confirm the bacteria is Kan resistant (thus the plasmid successfully inserted) and to isolate the colony and show it can grow on different lept. A maximum of four colonies per plate were chosen and labeled: A1, B1-4, C1-4, D1-4, E1-4, F1, G1-3, H1-2. They were all plated onto both types of media along with a WT on the final plate.