01/20/10
- ✓ PCR: KAH94, 95/JDS48/49 minipreps from 1/19
- ✓ Minipreps: KAH139, 140, 141 (2 each); KAH118/MV2(12)
- ✓ Assemblies: KAH139, 140, 141/ V0201
Minipreps
> Check with E/P digests
Reagent |
1-6 |
7-18
|
Expected: V0120 = 3200 MV2 = 4529 1,2. KAH139 = 2945 3,4. KAH140 = 2939 5,6. KAH141 = 2527 7-18. KAH118/MV2 = 7930
|
15 μL/lane; 1% agarose
|
DNA(plasmid) |
2.0 |
4.0
|
10X buffer |
1.5 |
1.5
|
EcoRI |
1.0 |
1.0
|
PstI |
1.0 |
1.0
|
dH2O |
9.5 |
7.5
|
|
15 μL |
15 μL --> 37°C/ ~15 min.
|
PCR
--> Check KAH94, 95/JDS48/49 minipreps from 1/19 (24 total)
--> Primers: RFP1 (used for RFP expression RT-PCR; should also work on DNA)
Reagent |
Volume |
|
Expected: 1-6. KAH94/JDS49 = 100 7-12. KAH95/JDS49 = 100 13-18. KAH94/JDS48 = 100 19-24. KAH95/JDS48 = 100 25. KAH94/MV2 = 100 26. MV5 (NLS-pcDNA) = 0
|
10 μL/lane, 1% agarose
|
DNA (plasmid) |
0.5
|
10 μM primer mix |
1.0
|
2x GoTaq Green |
10.0
|
dH2O |
8.5
|
|
20 μL
|
--> PCR (96-well): 95°C/ 3 min.; [95°C/ 30 sec., 57°C/ 30 sec., 72°C/ 30 sec] x30; 72°C/ 3 min.
--> Still unclear. Tomorrow digest w/ BglII
Assemblies
- KAH139/V0201: (1) KAH139/(N/S)/2945 + (4) V0201/(N/S)/5157
- KAH140/V0201: (2) KAH140/(N/S)/2939 + "
- KAH141/V0201: (3) KAH141/(N/S)/2527 + "
> Digests (Fermentas FD)
--> Use ScaI to cut V0120 for KAH139, 140
Reagent |
1,2 |
1,2
|
30 μL/lane, 1% agarose
|
DNA (plasmid) |
10.0 |
10.0
|
10x buffer |
3.0 |
3.0
|
NotI |
1.0 |
1.0
|
SpeI |
1.0 |
1.0
|
ScaI |
1.0 |
---
|
dH2O |
15.0 |
15.0
|
|
31 μL |
30 μL --> 37°C/ ~30 min.
|
> Measure conc.'s
Sample |
OD260 |
260/280 |
ng/μL
|
1. KAH139(N/S) |
0.350 |
2.03 |
17.5
|
2. KAH140(N/S) |
0.443 |
1.81 |
22.2
|
3. KAH141(N/S) |
0.451 |
2.02 |
22.5
|
4. V0201(N/S) |
0.536 |
1.87 |
26.8
|
> Ligations
Ligation |
Plate results (lig : neg crtl) 01/21/10
|
1. KAH139(N/S)/2945, 29 ng + V0201(N/S)/5127, 25 ng |
KAH139/V0201 10:1 (Pick 2)
|
2. KAH140(N/S)/2939, 29 ng + " |
KAH140/V0201 10:1 (Pick 2)
|
3. KAH141(N/S)/2527, 25 ng + " |
KAH141/V0201 10:1 (Pick 2)
|
4. V0201(N/S)/ 25 ng |
|
|
1 |
2 |
3 |
4
|
Insert DNA |
2.0 |
2.0 |
2.0 |
---
|
Vector DNA |
1.0 |
1.0 |
1.0 |
1.0
|
2x lgn buf (Roche) |
5.0 |
5.0 |
5.0 |
5.0
|
T4 ligase (NEB) |
1.0 |
1.0 |
1.0 |
1.0
|
dH2O |
1.0 |
1.0 |
1.0 |
3.0
|
|
10μL |
10μL |
10μL |
10μL --> R.T./ 10 min.; Add 20 μL Z-DH5α
|
|