07/17/2015
Re-try LasR_MRV gel extraction:
| Reagent
|
Volume (uL)
|
| Vector |
15
|
| 10x Buffer |
3
|
| BbsI |
1
|
| Water |
11
|
| Total: |
30
|
- Run in 0.8% gel (0.48g agarose to 60mL TAE Buffer), at 90 volts for 1 hour.
Digest EsaInvRep_MRV with SAP, EcoRI, and XbaI for regulator insertion.
| Reagent
|
Volume (uL)
|
'
|
| Vector |
12.8 |
1ug at 78ng/uL
|
| 10x Buffer |
3 |
|
| EcoRI |
1 |
|
| XbaI |
1 |
|
| Phosphatase |
1 |
|
| Water |
11.2 |
|
| Total |
30 |
|
- Incubate at 37°C for 30 minutes, then heat deactivate.
- EcoRI and XbaI deactivation temperatures are: 80°C for 5 minutes and 65°C for 20 minutes.
Digest EsaR PCR-purified PCR product with EcoRI and XbaI to prep for ligation
| Reagent
|
Volume (uL)
|
| DNA (200ng) |
3.6
|
| 10x Buffer |
3
|
| EcoRI |
1
|
| XbaI |
1
|
| Water |
21.4
|
| Total |
30
|
- Incubate at 37°C for 15 minutes, then heat deactivate.
- EcoRI and XbaI deactivation temperatures are: 80°C for 5 minutes and 65°C for 20 minutes.
Send pLux1, 2, 3 out for sequencing.
- Used RM inv_rep F primer (Ryan didn't create a reverse).
- I sent pLux 1 out as well because I am curious as to why there are three bands.
|
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