11/19/09
- ✓ Minipreps: KAH77(2), 78(3), 79(3), 83(2), 84(2), 85(2)
- ✓ Assemblies: NLS myc stop (KAH011), K-stop (KAH012); Postpone ---> KAH93, 94, 95
- ✓ Cultures: KAH86(2), 87(2), 88(2), 89(2), 90(2), 91(2), 92(2); KAH84(2, re-pick); 5 ml ea.
Minipreps
> Check with E/P digests (Fermentas FD)
Reagent |
Volume
|
Expected: 1,2. KAH77 = 3480 3-5. KAH78 = 3652 6,7. KAH79 = 3724 8,9. KAH83 = 443 10,11. KAH84 = 615 12,13. KAH85 = 687
|
|
DNA(plasmid) |
2.0 μL
|
10X buffer |
1.5
|
EcoRI |
1.0
|
PstI |
1.0
|
dH2O |
9.5
|
|
15 μL --> 37°C/ ~15 min.
|
Note: Re-do KAH77-79 digests tomorrow, triple digest to cut V0120
Assemblies
- KAH011: NLS-myc-stop/(X/P ds-oligo)/64 + V0120/(X/P)/3200
- KAH012: Kstop/(X/P ds-oligo)/24 + V0120/(X/P)/3200
(tomorow)
- KAH93: 2xHS4-Ubc-K-Gal4DB-PEST-2xmCh-NLS-stop (KAH77)/(E/S)/3480 + 5xLuc target-polyA-2xHS4 (KAH67)/(E/X)/4553
- KAH94: 2xHS4-HPK-K-Gal4DB-PEST-2xmCh-NLS-stop (KAH78)/(E/S)/3652 + "
- KAH95: 2xHS4-CMV-K-Gal4DB-PEST-2xmCh-NLS-stop (KAH79)/(E/S)/3724 + "
> Ligations
Ligation |
Plate results (lig : neg crtl) 11/20/09
|
1. NLS-myc-stop(X/P ds-oligo)/64, > 0.8 ng + V0120(X/P)/3200, 20 ng |
KAH011, 0 colonies (re-do)
|
2. Kstop(X/P ds-oligo)/24, > 0.3 ng + V0120(X/P)/3200, 20 ng |
KAH012, 1 colony (re-do)
|
3. V0120/(X/P)/ 20 ng |
0 colonies
|
|
1 |
2 |
3 |
|
Insert DNA |
0.2 |
0.2 |
---
|
Vector DNA (from Mara) |
2.5 |
2.5 |
2.5
|
2X lgn buf (Roche) |
5.0 |
5.0 |
5.0
|
T4 ligase (NEB) |
1.0 |
1.0 |
1.0
|
dH2O |
1.3 |
1.3 |
1.5
|
|
10 μL |
10 μL |
10 μL --> R.T./ ~15 min.; Add 25μL Zymo DH5α
|
|