07/25/11
- ✓ Retransformations: 183-DF4, -DF5, -DF6/MV8; 183-177/MV8; STOP
- ✓ Assemblies A: DPRE-208/V0200; KAH225, KAH226
- ✓ Assemblies B: miR-luc sensor "198/MV8" set
Assemblies
- DPRE-208/V0200: (1) DPRE/(N)/1740 + (2) KAH208/V0200/(N dp)/6326
- KAH225: mCh/(S/P)/705 ✓ + (3) p65/(X/P)/783
> Digests (Fermentas FD)
| Reagent |
Volume |
|
|
| DNA (plasmid) |
up to 25 μL
|
| 10x buffer |
3.0
|
| enzyme 1 |
1.0
|
| enzyme 2 |
1.0
|
| dH2O |
---
|
| |
30 μL --> 37°C/ ~30 min.
|
> Measure conc.'s
| Sample |
OD260 |
260/280 |
ng/μL
|
| 1. DPRE (N) |
1.518 |
1.83 |
75.9
|
| 2. KAH208/V0200 (N) |
0.385 |
1.59 |
19.2
|
| 3. p65 (X/P) |
0.359 |
1.56 |
18.0
|
> Dephosphorylation (Roche)
| Reagent |
Volume
|
| DNA (clean digest) |
up to 17 μL (max, 326.4 ng)
|
| 10x buffer d.p. |
2.0
|
| phosphatase |
1.0
|
| dH2O |
---
|
| |
20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 16 ng/μL
|
> Ligations
| Ligation |
Plate results (lig : neg crtl) mm/dd/yy
|
| 1. DPRE(N)/1740, 11 ng + KAH208/V0200(N dp)/6326, 20 ng |
DPRE-208/V0200 >10:1 (Pick 4)
|
| 2. KAH208/V0200(N dp)/ 20 ng |
|
| 3. p65(X/P)/783, 8 ng + mCh(S/P)/~3950, 20 ng |
KAH225 >10:1 (Pick 2)
|
| 4. mCh(S/P)/ 20 ng |
|
| |
1 |
2 |
3 |
4 |
|
| Insert DNA |
0.2 |
--- |
0.4 |
---
|
| Vector DNA |
1.3 |
1.3 |
0.4 |
0.4
|
| 2x lgn buf (Roche) |
5.0 |
5.0 |
5.0 |
5.0
|
| T4 ligase (NEB) |
1.0 |
1.0 |
1.0 |
1.0
|
| dH2O |
2.5 |
2.7 |
3.2 |
3.6
|
| |
10 μL |
10 μL |
10 μL |
10 μL |
|
Assemblies B
--> Last time (6/06/11), may have used too much insert. Use calculated amount this time
--> See 7/20/11 for 198/MV8 digest and concentration
- KAH186-196/MV8: KAH186/(E/S)/2025 ✓ + KAH198/MV8(E/X)/7978 ✓
- KAH184-196/MV8: KAH187/(E/S)/2269 ✓ + "
- KAH187-196/MV8: KAH187/(E/S)/2269 ✓ + "
- KAH188-196/MV8: KAH188/(E/S)/2124 ✓ + "
- KAH189-196/MV8: KAH189/(E/S)/2296 ✓ + "
- KAH190-196/MV8: KAH190/(E/S)/2368 ✓ + "
- KAH191-196/MV8: KAH191/(E/S)/2223 ✓ + "
- KAH183-196/MV8: KAH191/(E/S)/2223 ✓ + "
- KAH192-196/MV8: KAH192/(E/S)/2461 ✓ + "
> Ligations
| Ligation |
Plate results (lig : neg crtl) 07/14/11
|
| 1-9. KAH###(E/S)/~2200, 11 ng + KAH198/MV8(E/X)/8050, 20 ng |
Notes: ~5:1 for all plates
|
| 10. KAH198/MV8(E/X)/ 20 ng |
|
| |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
|
| Insert DNA |
1.0 |
1.0 |
1.5 |
1.0 |
1.0 |
0.5 |
1.0 |
0.5 |
0.8 |
---
|
| Vector DNA |
0.4 |
0.4 |
0.4 |
0.4 |
0.4 |
0.4 |
0.4 |
0.4 |
0.4 |
0.4
|
| 2x lgn buf (Roche) |
5.0 |
5.0 |
5.0 |
5.0 |
5.0 |
5.0 |
5.0 |
5.0 |
5.0 |
5.0
|
| T4 ligase (NEB) |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0
|
| dH2O |
2.6 |
2.6 |
2.1 |
2.6 |
2.6 |
3.1 |
2.6 |
3.1 |
2.8 |
3.6
|
| |
10 μL |
10 μL |
10 μL |
10 μL |
10 μL |
10 μL |
10 μL |
10 μL |
10 μL |
10 μL
|
--> Add to 30 μL DH5α Turbo; plate on 100 μg/mL amp
|
Project name
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