"
Team:Cambridge/Signalling/Lab Work
From 2008.igem.org
(→Agr A and C ligation to pSB4C5) |
(→August, 16th) |
||
| Line 14: | Line 14: | ||
The bands of appropriate sizes were extracted for transformation. | The bands of appropriate sizes were extracted for transformation. | ||
| + | =August, 18th= | ||
| + | ==Check promoters== | ||
| + | On Friday, PCR out promoters, we want to check them. | ||
| + | - Run a gel | ||
| + | |||
| + | - Results : good size of bands!!! | ||
| + | |||
| + | ==Transformation of AgrA and AgrC== | ||
| + | |||
| + | AgrA and C gell-extracted ligation was transformed into Top 10 cells using standard protocol. Puc9 was used as a positive control. | ||
| + | |||
| + | =August, 19th= | ||
| + | |||
| + | |||
| + | ==Lux parts== | ||
| + | |||
| + | To make the Lux Receiver, we need 4 different parts ; | ||
| + | |||
| + | * R0040, TetR repressible promoter | ||
| + | |||
| + | * SO168, luxR + double terminator | ||
| + | |||
| + | * R0062, promoter activated by luxR | ||
| + | |||
| + | * JO4630 (GFP + double terminator) | ||
| + | |||
| + | All these parts have been transformed into E.coli. We want to test them. R004, R0062 and JO4630 have already been tested, it should be fine. | ||
| + | We received from the MIT R0040, R0062 and S068 already transformed into E.coli, so we want to check these stocks (which are certainly fine) and use them. | ||
| + | For JO4630, we want to double check our transformation. | ||
| + | |||
| + | - Plate on antibiotic plates and do LB stocks of single colony from the MIT stock (R0040, R0062 and S0168). | ||
| + | |||
| + | - Put on Kan plates 4 different colonies from J04630 (transformation Amp plate) and also incubate these colonies into LB | ||
<!-- ## Do not edit below this line unless you know what you are doing. ## --> | <!-- ## Do not edit below this line unless you know what you are doing. ## --> | ||
Revision as of 17:32, 28 October 2008
August, 16th
Agr A and C ligation to pSB4C5
Ligation was performed using Fermentas Rapid Ligation kit according to their protocol.
After ligation, samples were visualized on a gel, but little product of the correct size was seen.
For agrA we should have gotten a band of size 3700 (3000plasmid + 700 insert)
For agrC we should have gotten a band of size 4300 (3000plasmid + 1300insert)
The bands of appropriate sizes were extracted for transformation.
August, 18th
Check promoters
On Friday, PCR out promoters, we want to check them.
- Run a gel
- Results : good size of bands!!!
Transformation of AgrA and AgrC
AgrA and C gell-extracted ligation was transformed into Top 10 cells using standard protocol. Puc9 was used as a positive control.
August, 19th
Lux parts
To make the Lux Receiver, we need 4 different parts ;
- R0040, TetR repressible promoter
- SO168, luxR + double terminator
- R0062, promoter activated by luxR
- JO4630 (GFP + double terminator)
All these parts have been transformed into E.coli. We want to test them. R004, R0062 and JO4630 have already been tested, it should be fine. We received from the MIT R0040, R0062 and S068 already transformed into E.coli, so we want to check these stocks (which are certainly fine) and use them. For JO4630, we want to double check our transformation.
- Plate on antibiotic plates and do LB stocks of single colony from the MIT stock (R0040, R0062 and S0168).
- Put on Kan plates 4 different colonies from J04630 (transformation Amp plate) and also incubate these colonies into LB
