February 8, 2013
- Assembly: 5xGal4 : [Spacer : HSVtk TATA] : K : AmCyan : AmCyan : NLS : STOP : [polyA2] KAH201/KAH182; AmCyan reporter gene for chromatin proteins.
Assemblies
- KAH201/KAH182: (1) KAH201/(S/P)/203 + (2) KAH182/(X/P)/1675
> Digests (Fermentas FD)
Reagent |
Volume |
|
30 μL/lane, 1% agarose; Ladder
|
DNA (plasmid) |
up to 25 μL
|
10x buffer |
3.0
|
enzyme 1 |
1.0
|
enzyme 2 |
1.0
|
dH2O |
---
|
|
30 μL --> 37°C/ ~15 min.
|
> Measure conc.'s of gel purified fragments
Sample |
OD260 |
260/280 |
ng/μL
|
1. KAH201 (S/P) |
TBD |
TBD |
TBD
|
2. KAH182 (X/P) |
TBD |
TBD |
TBD
|
> Ligations
Ligation |
Plate results (lig : neg crtl) 11/31/12
|
1. KAH201 (S/P)/203, 8 ng + KAH182 (X/P)/1675, 25 ng |
KAH3/MV2 >10:1 (Pick 2)
|
2. KAH182 (X/P)/ 25 ng |
zero colonies
|
|
1 |
2 |
|
Insert DNA |
0.3 |
--- |
|
Vector DNA |
0.3 |
0.3 |
|
2x lgn buf (Roche) |
5.0 |
5.0 |
|
T4 ligase (NEB) |
1.0 |
1.0 |
|
dH2O |
3.4 |
3.7 |
|
|
10 μL |
10 μL |
|
- Follow quick transformation protocol
- Add 30 uL DH5α-Turbo to each ligation; plate on 100 μg/mL Amp agar
|