12/14/09
- ✓ Minipreps: digests for 12/12/09 minipreps
- ✓ Cultures: KAH123(3), 124(3), 125(3)
- ✓ Assemblies: KAH126-133 into MV2
Minipreps
> Check with E/P digests
Reagent |
Volume
|
Expected: 1,2. KAH126 = 1880 3,4. KAH127 = 1880 5,6. KAH128 = 1880 7,8. KAH129 = 1880 9,10. KAH130 = 2864 11,12. KAH131 = 2867 13,14. KAH132 = 1688 15,16. KAH133 = 1532 17,18. KAH134 = 1400
|
15 μL/lane; 1% agarose
|
DNA(plasmid) |
2.0 μL
|
10X buffer |
1.5
|
EcoRI |
1.0
|
PstI |
1.0
|
dH2O |
9.5
|
|
15 μL
|
--> 37°C/ ~15 min.
Assemblies
- KAH126/MV2: (1) KAH126/(E/P)/1880 + MV2/(E/P)/4529 ✓
- KAH127/MV2: (2) KAH127/(E/P)/1880 + "
- KAH128/MV2: (3) KAH128/(E/P)/1880 + "
- KAH129/MV2: (4) KAH129/(E/P)/1880 + "
- KAH130/MV2: (5) KAH130/(E/P)/2864 + "
- KAH131/MV2: (6) KAH131/(E/P)/2867 + "
- KAH132/MV2: (7) KAH132/(E/P)/1688 + "
- KAH133/MV2: (8) KAH133/(E/P)/1532 + "
> Digests (Fermentas FD)
--> Triple digest KAH130 & 131 w/ E/P/ScaI
Reagent |
Vol (1-4, 7, 8) |
Vol (5, 6)
|
30 μL/lane, 1% agarose
|
DNA (plasmid) |
15.0 |
15.0
|
10x buffer |
3.0 |
3.0
|
enzyme 1 |
1.0 |
1.0
|
enzyme 2 |
1.0 |
1.0
|
ScaI |
--- |
1.0
|
dH2O |
10.0 |
9.0
|
|
30 μL |
30 μL --> 37°C/ ~30 min.
|
> Measure conc.'s
Sample |
OD260 |
260/280 |
ng/μL
|
1. KAH126(E/P) |
0.705 |
1.86 |
35.2
|
2. KAH127(E/P) |
1.095 |
1.88 |
54.7
|
3. KAH128(E/P) |
0.916 |
1.88 |
45.8
|
4. KAH129(E/P) |
0.892 |
1.85 |
44.6
|
5. KAH130(E/P) |
0.776 |
2.02 |
38.8
|
6. KAH131(E/P) |
0.620 |
1.93 |
31.0
|
7. KAH132(E/P) |
0.729 |
1.88 |
36.5
|
8. KAH133(E/P) |
0.380 |
1.96 |
19.0
|
> Ligations
Ligation |
Plate results (lig : neg crtl) 12/15/09
|
1. KAH126(E/P)/1880, 21 ng + MV2/(E/P)/4529, 25 ng |
KAH126/MV2 10:1 (Pick 2)
|
2. KAH127(E/P)/1880, 21 ng + " |
KAH127/MV2 10:1 (Pick 2)
|
3. KAH128(E/P)/1880, 21 ng + " |
KAH128/MV2 10:1 (Pick 2)
|
4. KAH129(E/P)/1880, 21 ng + " |
KAH129/MV2 10:1 (Pick 2)
|
5. KAH130(E/P)/2864, 32 ng + " |
KAH130/MV2 10:1 (Pick 2)
|
6. KAH131(E/P)/2867, 32 ng + " |
KAH131/MV2 10:1 (Pick 2)
|
7. KAH132(E/P)/1688, 19 ng + " |
KAH132/MV2 10:1 (Pick 2)
|
8. KAH133(E/P)/1532, 17 ng + " |
KAH133/MV2 10:1 (Pick 2)
|
2. MV2(E/P)/ 25 ng |
|
|
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9
|
Insert DNA |
1.0 |
1.0 |
1.0 |
1.0 |
1.5 |
1.5 |
1.0 |
1.0 |
---
|
Vector DNA |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0 |
1.0
|
2x lgn buf (Roche) |
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
|
dH2O |
2.0 |
2.0 |
2.0 |
2.0 |
1.5 |
1.5 |
2.0 |
2.0 |
3.0
|
|
10μL |
10μL |
10μL |
10μL |
10μL |
10μL |
10μL |
10μL |
10μL --> R.T./ ~10 min.; Add 20μL Z-DH5α
|
|