February 8, 2013
- Assembly: (5xGal4 : [Spacer : HSVtk TATA]) : (K : AmCyan : AmCyan : NLS : STOP : [polyA2]) (KAH201)/(KAH182); AmCyan reporter gene for chromatin proteins.
Assemblies
- Note, KAH201 is in vector V0120, so add 3200 bp to the size.
- KAH201/KAH182: (1) KAH201/(S/P)/203+3200 + (2) KAH182/(X/P)/1675
> Digests (Fermentas FD)
Reagent |
Volume |
|
30 μL/lane, 1% agarose; Ladder
|
DNA (plasmid) |
20 μL
|
10x buffer |
3.0
|
enzyme 1 |
1.0
|
enzyme 2 |
1.0
|
dH2O |
5.0 μL
|
|
30 μL --> 37°C/ ~15 min.
|
> Measure conc.'s of gel purified fragments
Sample |
OD260 |
260/280 |
ng/μL
|
1. KAH201 (S/P) |
0.048 |
1.88 |
48.1
|
2. KAH182 (X/P) |
0.008 |
1.93 |
8.1
|
> Ligations (corrected version, 3/04/13)
Ligation |
Plate results (lig : neg crtl) 02/20/13
|
1. KAH201 (S/P)/3403, 25 ng + KAH182 (X/P)/1675, 26 ng |
0
|
2. KAH201 (X/P)/3403, 25 ng |
0
|
|
1 |
2 |
|
Insert DNA (KAH182) |
0.74 |
--- |
|
Vector DNA (KAH201) |
0.52 |
0.52 |
|
2x lgn buf (Roche) |
5.0 |
5.0 |
|
T4 ligase (NEB) |
1.0 |
1.0 |
|
dH2O |
2.74 |
3.48 |
|
|
10 μL |
10 μL |
|
- Follow quick transformation protocol
- Add 30 uL DH5α-Turbo to each ligation; plate on 100 μg/mL Amp agar
- No colonies were formed, this will be repeated. See 2/26/13 entry.
|