Buckner Lab:Notebook/Summer 2010/2010/05/17: Difference between revisions
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==Experimental Notes== | ==Experimental Notes== | ||
-Follow LB agar recipe | ''LB and LB+Amp agar Plates'' | ||
-Make sure that the agar mixes fully in the LB solution. The batch today did not. Less agar can result in softer plates. | -Follow LB agar recipe<br> | ||
-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. | -Make sure that the agar mixes fully in the LB solution. The batch today did not. Less agar can result in softer plates.<br> | ||
-One marker stripe means LB only. Two markers stripes means LB and Amp. | -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.<br> | ||
-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. | -One marker stripe means LB only. Two markers stripes means LB and Amp.<br> | ||
-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. <br> | |||
''Plasmid Recombination''<br> | ''Plasmid Recombination''<br> | ||
-Retrieved E. coli JM109 from -80C freezer and thawed on ice (~15 min) | -Retrieved E. coli JM109 from -80C freezer and thawed on ice (~15 min)<br> | ||
-Pipette 50uL of cells into the 0.5uL plasmid DNA (Dr. JB already ligated the plasmids) Put tube on ice for 20 min. | -Pipette 50uL of cells into the 0.5uL plasmid DNA (Dr. JB already ligated the plasmids) Put tube on ice for 20 min.<br> | ||
-Heat shocked each tube simultaneously at 42C for 46 sec, then moved to the ice for another 2 min. | -Heat shocked each tube simultaneously at 42C for 46 sec, then moved to the ice for another 2 min.<br> | ||
-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. | -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.<br> | ||
-Plated stock bacteria (DH5a, HB101, TG1) on LB agar plates and incubate at 35C (12:41 pm) | -Plated stock bacteria (DH5a, HB101, TG1) on LB agar plates and incubate at 35C (12:41 pm)<br> | ||
-Plated fas1b, KER 1D, Kn1. 2 plates per plasmid: 1 25uL, 1 100uL. Incubated 35C (1:30pm) | -Plated fas1b, KER 1D, Kn1. 2 plates per plasmid: 1 25uL, 1 100uL. Incubated 35C (1:30pm)<br> | ||
BBond | BBond |
Revision as of 12:17, 17 May 2010
LB agar and Recombination | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> |
Plates and PlasmidsObjectives:
Experimental NotesLB and LB+Amp agar Plates
-Follow LB agar recipe Plasmid Recombination BBond |