02/22/11
- ✓ Gaussia luciferase: check minipreps with NotI digestion
- ✓ Biobrick PCR: G luc
Gaussia luc minipreps
> Check with NotI digest
| Reagent |
Volume
|
Expected:
Vector = 3030
1-6. G luc/ Addgene22522 = 1433
|
15 μL/lane; 1% agarose
|
| DNA(plasmid) |
2.0 μL
|
| 10X buffer |
1.5
|
| NotI |
1.0
|
| dH2O |
10.5
|
| |
15 μL --> 37°C/ ~15 min.
|
BioBrick PCR
--> Gaussia luciferase from Addgene22522 template
- KAH016: (1) Gluc/(X/P PCR)/558 + V0120/(X/P)/~3200 ✓
> PCR
--> Primers: BB_Gluc f/ BB_Gluc r
| Reagent |
Volume
|
| DNA (plasmid) |
1.0
|
| 10 μM primers |
1.0
|
| 2x GoTaq |
25.0
|
| dH2O |
23.0
|
--> BioRad DNA Engine, block A
- 95°C/ 3 min.
- [95°C/ 30 sec; 55°C/ 30 sec; 72 °C/ 1 min.] x30
- 72 °C/ 3 min.
- 4 °C/ ∞
> Zymo clean PCR; elute w/ 25 μL dH2O
> Digests (Fermentas FD)
| Reagent |
Volume |
|
30 μL/lane, 1% agarose
Expected = 558 bp
|
| PCR DNA |
25.0
|
| 10x buffer |
3.0
|
| XbaI |
1.0
|
| PstI |
1.0
|
| dH2O |
---
|
| |
30 μL --> 37°C/ ~30 min.
|
> Measure conc.'s
| Sample |
OD260 |
260/280 |
ng/μL
|
| 1. G luc (X/P) |
### |
### |
###
|
??? -- Nanodrop computer isn't working. Just use max volume insert for 10.0 μL reaction.
> Ligations
| Ligation |
Plate results (lig : neg crtl) 02/23/11
|
| 1. G luc(X/P)/558, 8.7 ng + V0120(X/P)/~3200, 25 ng |
G luc (KAH016) >10:1 (Pick 4)
|
| 2. V0120(X/P)/ 25 ng |
|
| |
1 |
2
|
| Insert DNA |
3.0 |
---
|
| Vector DNA |
1.0 |
1.0
|
| 2x lgn buf (Roche) |
5.0 |
5.0
|
| T4 ligase (NEB) |
1.0 |
1.0
|
| dH2O |
--- |
3.0
|
| |
10 μL |
10 μL
|
--> R.T./ ~15 min.; Add to 30 μL DH5α turbo
| 
Project name
