07/23/2015
- pEsa_MRV + EsaR had a lot of colonies; backbone controls had fewer colonies.
- LasR_MRV + pLas has lots of colonies, but its backbones had many as well.
Colony PCR for pEsa.
| Reagent
|
Vol (uL)
|
Master Mix (x20)
|
| F Primer |
0.5 |
10
|
| R Primer |
0.5 |
10
|
| DNA |
- - |
- -
|
| GoTaq MM |
12.5 |
250
|
| Water |
11.5 |
230
|
| Total: |
25 |
500
|
- F Primer - RM seqver mod receiver LuxR home
- R Primer - RM seqver regulator R
- 8 colonies were picked for LasR_MRV + pLas ligation
- 4 colonies were picked for the backbones (4 for ligation and 4 for no ligation)
 
Prepare liquid cultures for LasR to digest verify tomorrow. (picked 6 colonies)
Pick more colonies from Dr. Haynes' pAub plate (picked 4 colonies)
Prepare pLux_MRV, LuxR, for ligation
- pLux_MRV digestion -
| Reagent
|
Volume (uL)
|
'
|
| Vector |
7.8 |
1ug at 127.42ng/uL
|
| 10x Buffer |
3 |
|
| EcoRI |
1 |
|
| XbaI |
1 |
|
| Phosphatase |
1 |
|
| Water |
16.2 |
|
| Total |
30 |
|
- LuxR digestion (already PCR purified)
| Reagent
|
Volume (uL)
|
'
|
| DNA |
5.9 |
200ng at 34ng/uL
|
| 10x Buffer |
3 |
|
| EcoRI |
1 |
|
| XbaI |
1 |
|
| Water |
19.1 |
|
| Total |
30 |
|
- Both: Incubate at 37°C for 15 minutes, then heat deactivate.
- Heat deactivation for E + X is - 80°C for 5 minutes, 65°C for 20 minutes
|
Project name
Main project page
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