Buckner Lab:Notebook/Summer 2010/2010/05/17: Difference between revisions
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*Plate Recombinant E. coli | *Plate Recombinant E. coli | ||
==Experimental Notes== | |||
-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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Revision as of 12:16, 17 May 2010
 LB agar and Recombination
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Plates and PlasmidsObjectives:
Experimental Notes-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 | |
