05/01/15
- Assembly: KAH87 (hPcTF ORF) into MV10
Assembly: KAH87 (hPcTF ORF) into MV10
- Minipreps (5 mL cultures)
- Sigma GenElute kit
- Elute w/ 75 μL elution sln.
- KAH87
- MV10
- Measure conc.'s of minipreps
Sample |
OD260 |
260/280 |
ng/μL
|
1. KAH87 (X/S) |
0.180 |
1.923 |
179.8
|
2. MV10 (X) |
0.371 |
1.926 |
370.5
|
- KAH87/MV10: KAH87/(X/S)/1053 + MV10/(X)/5197
- KAH87, X/S
- MV10, X
Reagent
|
Rxn 1
|
Rxn 2
|
|
DNA (plasmid) |
20.0 |
15.0
|
10x buffer |
3.0 |
3.0
|
XbaI |
1.0 |
1.0
|
SpeI |
1.0 |
---
|
dH2O |
5.0 |
10.0
|
|
30 μL
|
--> 37°C/ ~30 min.
- Gel purify
- Sigma kit
- Elute & back-elute w/ 25 μL elution sln.
Sample |
OD260 |
260/280 |
ng/μL
|
1. KAH87 (X/S) |
0.003 |
1.571 |
3.49
|
2. MV10 (X) |
0.070 |
1.843 |
70.41
|
- Dephosphorylation (Roche)
- Dephos the MV10 (X) fragment
Reagent
|
Volume
|
DNA (clean digest) |
7.1 (500 ng)
|
10x buffer d.p. |
2.0
|
phosphatase |
1.0
|
dH2O |
9.9
|
|
20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
|
- Ligations
- 2:1 ratio calc.: 1053 (KAH87) / 5197 (MV10) * 2 * 50 = 20 ng KAH87
- KAH87 (X/S) + MV10 (X)
- MV10 (X dp), neg ctrl.
|
1 |
2 |
|
Insert DNA |
5.7 |
--- |
|
Vector DNA |
2.0 |
2.0 |
|
2x lgn buf (Roche) |
8.7 |
8.7 |
|
T4 ligase (NEB) |
1.0 |
1.0 |
|
dH2O |
--- |
5.7 |
|
|
17.4 μL |
17.4 μL |
|
|