Haynes Lab:Notebook/Characterizing AHL quorum sensing homologs/2013/03/21: Difference between revisions
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==Entry title== | ==Entry title== | ||
Picked 2 E0240 colonies into 2ml. Checked the plates that I pipetted S-R pairs onto last night under UV, didn't see anything. | |||
Pipetted the co-cultures onto the plate (15-20ul). Didn't see anything. | |||
Miniprepped at 6ish. low yields. Cut E0240 with X/P, ran on gel. | |||
Grew from 11am-6 or 7pm. Spun down co-cultures at 12.5ish for 2mins. Pours off supernatant, resuspended in ~100ul H2) (ended up being ~150 total with residual LB and cells). Used plate reader to check fluorescence for CFP and got higher readings for RhlI-RhlR. | |||
{| {{table}} | |||
|- | |||
|RhlR only||LuxI-RhlR||LasI-RhlR||RhlI-RhlR | |||
|- | |||
|40515||39818||40325||47768 | |||
|- | |||
|} | |||
<br> | |||
ligation of K092900-E0240 | |||
{| {{table}} | |||
|- | |||
|K092900||4ul of 12ng/ul | |||
|- | |||
|E0240|| 14ul (didn't check conc, was faint on gel) | |||
|- | |||
|2x buffer||20ul | |||
|- | |||
|T4 ligase|| 1ul | |||
|- | |||
|} | |||
Transformed into 50ul of BL21. At RT for 10min, ice for 13min, 42deg for 45s | |||
<br> | |||
New experiment: grow separate, add sups, let grow. measure at different time points. 1ml for each measurement. Will need to spin will to remove all sender cells. Run in triplicate. | |||
{| {{table}} | |||
|- | |||
| || LuxI||LasI||RhlI||RhlR only | |||
|- | |||
|1hr|| || || || | |||
|- | |||
|3hr || || || || | |||
|- | |||
|5hr || || || || | |||
|- | |||
|7hr || || || || | |||
|- | |||
|} | |||
4-6ml cultures of senders | |||
3-35ml cultures of RhlR | |||
Started at 11:30PM | |||
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Revision as of 13:01, 28 May 2013
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Entry titlePicked 2 E0240 colonies into 2ml. Checked the plates that I pipetted S-R pairs onto last night under UV, didn't see anything. Pipetted the co-cultures onto the plate (15-20ul). Didn't see anything. Miniprepped at 6ish. low yields. Cut E0240 with X/P, ran on gel. Grew from 11am-6 or 7pm. Spun down co-cultures at 12.5ish for 2mins. Pours off supernatant, resuspended in ~100ul H2) (ended up being ~150 total with residual LB and cells). Used plate reader to check fluorescence for CFP and got higher readings for RhlI-RhlR.
Transformed into 50ul of BL21. At RT for 10min, ice for 13min, 42deg for 45s
4-6ml cultures of senders 3-35ml cultures of RhlR Started at 11:30PM |