Buckner Lab:Notebook/Summer 2010/2010/05/17

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LB agar and Recombination Main project page
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Plates and Plasmids

Objectives:

  • Pour LB and LB + Amp agar plates
  • Plate Stock Bacteria (DH5a, HB101, TG1)
  • Insert Dr. JB plasmid DNA into E. coli JM109's
  • Plate Recombinant E. coli

Experimental Notes

LB and LB+Amp agar Plates
-Follow LB agar recipe
-Make sure that the agar mixes fully in the LB solution. The batch today did not. Less agar can result in softer plates.
-After autoclaving, allow LB mixture to cool to ~60C and pour into plates. Use a paper towel around the edge to catch the "drip" from pouring. Don't overfill plates. Half of the solution should be LB agar and the other half LB + Amp agar.
-One marker stripe means LB only. Two markers stripes means LB and Amp.
-Placed 125uL Amp into ~250mL of the LB (half of the 500mL I made) and poured 5 plates before realizing that the solution needed 250uL Amp total. I added the additional Amp and noted the first five plates with an orange streak.

Plasmid Recombination
-Retrieved E. coli JM109 from -80C freezer and thawed on ice (~15 min)
-Pipette 50uL of cells into the 0.5uL plasmid DNA (Dr. JB already ligated the plasmids) Put tube on ice for 20 min.
-Heat shocked each tube simultaneously at 42C for 46 sec, then moved to the ice for another 2 min.
-450uL SOC was added to each tube, including the stock JM109's. Then each tube was placed in the holder and recovered in the incubator at 35-37C for 90min at 200 rotations/min.
-Plated stock bacteria (DH5a, HB101, TG1) on LB agar plates and incubate at 35C (12:41 pm)
-Plated fas1b, KER 1D, Kn1. 2 plates per plasmid: 1 25uL, 1 100uL. Incubated 35C (1:30pm)

BBond


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