Tokyo/Assay
From 2007.igem.org
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0:00 Start to prepare over night culture (1 tube for each) | 0:00 Start to prepare over night culture (1 tube for each) | ||
<br>12:00 Make fresh culture with 3 ul of the overnight culture in 3ml of LB + Amp + Kan (LBAK) (3 tubes for each) | <br>12:00 Make fresh culture with 3 ul of the overnight culture in 3ml of LB + Amp + Kan (LBAK) (3 tubes for each) | ||
| - | <br>Incubation in a shaker until the observed OD (ODobs) reaches up to 0.2 | + | <br> Incubation in a shaker until the observed OD (ODobs) reaches up to 0.2 |
| - | <br>Equalize ODobs of all the samples by adding LB media. | + | <br> Equalize ODobs of all the samples by adding LB media. |
<br>15:00 Centrifugation at 5000 rpm and wash out the LB media. | <br>15:00 Centrifugation at 5000 rpm and wash out the LB media. | ||
Revision as of 14:05, 19 October 2007
Contents |
AHL assay – hybrid promoter activation by endogenous AHL
Date
2007/10/18
Purpose
To check if worker E. coli (Sender) can produce enough AHL for our model to work by using different copy numbers of plasmids.
Samples
placI luxI on PSB1(high copy) and A4Δp(Sender 1)
placI luxI on PSB4(low copy) and A4Δp(Sender 2)
placI luxI on PBR322(low copy) and A4Δp(Sender 3)
(no promoter)tetR pBR322 and A4 Hybrid promoter(Receiver 1)
(no promoter)luxI pBR322 and A4Δp
Procedure
0:00 Start to prepare over night culture (1 tube for each)
12:00 Make fresh culture with 3 ul of the overnight culture in 3ml of LB + Amp + Kan (LBAK) (3 tubes for each)
Incubation in a shaker until the observed OD (ODobs) reaches up to 0.2
Equalize ODobs of all the samples by adding LB media.
15:00 Centrifugation at 5000 rpm and wash out the LB media.
Add cells with A4Δｐ and each of the following to 2.4 ml of LBAK
