12/10/09
- ✓ Minipreps: KAH109-116 (2 each, 16 total)
- ✓ Lab meeting at 4 pm! (Kevin and Karmella)
- ✓ Assemblies: KAH126-133; KAH134
- ✓ Cultures: KAH120-125 (3 each, 18 total)
Minipreps
> Check with E/P digests
Reagent |
Volume
|
Expected: 1,2. KAH109 = 1123 3,4. KAH110 = 1123 5,6. KAH111 = 1123 7,8. KAH112 = 1123 9,10. KAH113 = 2107 11,12. KAH114 = 2110 13,14. KAH115 = 931 15,16. KAH116 = 775
|
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: CMVTetO2-K(KAH3)/(S/P)/3951 ✓ + (1) KAH109/(X/P)/1123
- KAH127: " + (2) KAH110/(X/P)/1123
- KAH128: " + (3) KAH111/(X/P)/1123
- KAH129: " + (4) KAH112/(X/P)/1123
- KAH130: " + (5) KAH113/(X/P)/2107
- KAH131: " + (6) KAH114/(X/P)/2110
- KAH132: " + (7) KAH115/(X/P)/931
- KAH133: " + (8) KAH116/(X/P)/775
- KAH134: (10) Streptavidin tag(Joe)/(S/P)~3200 + (9) KAH67/(X/P)/1353
> Digests (Fermentas FD)
Reagent |
Volume |
|
30 μL/lane, 1% agarose
|
DNA (plasmid) |
up to 25 μL
|
10x buffer |
3.0
|
enzyme 1 |
1.0
|
enzyme 2 |
1.0
|
dH2O |
---
|
|
30 μL --> 37°C/ ~1 hr.
|
> Measure conc.'s
Sample |
OD260 |
260/280 |
ng/μL
|
1. KAH109(X/P) |
0.490 |
1.97 |
24.5
|
2. KAH110(X/P) |
0.444 |
2.06 |
22.2
|
3. KAH111(X/P) |
0.463 |
1.87 |
23.2
|
4. KAH112(X/P) |
0.474 |
1.94 |
23.7
|
5. KAH113(X/P) |
0.816 |
2.03 |
40.8
|
6. KAH114(X/P) |
0.748 |
2.11 |
37.4
|
7. KAH115(X/P) |
0.322 |
2.28 |
16.1
|
8. KAH116(X/P) |
0.295 |
2.28 |
14.7
|
9. KAH67(X/P) |
0.794 |
1.93 |
39.7
|
10. Strep(X/P) |
0.736 |
1.91 |
36.8
|
> Ligations
Ligation |
Plate results (lig : neg crtl) 12/11/09
|
1. KAH109(X/P)/1123, 11 ng + KAH03(S/P)/3951, 20 ng |
KAH126 >10:1 (Pick 2)
|
2. KAH110(X/P)/1123, 11 ng + " |
KAH127 >10:1 (Pick 2)
|
3. KAH111(X/P)/1123, 11 ng + " |
KAH128 >10:1 (Pick 2)
|
4. KAH112(X/P)/1123, 11 ng + " |
KAH129 >10:1 (Pick 2)
|
5. KAH113(X/P)/2107, 21 ng + " |
KAH130 >10:1 (Pick 2)
|
6. KAH114(X/P)/2110, 21 ng + " |
KAH131 >10:1 (Pick 2)
|
7. KAH115(X/P)/931, 9 ng + " |
KAH132 >10:1 (Pick 2)
|
8. KAH116(X/P)/775, 8 ng + " |
KAH133 >10:1 (Pick 2)
|
9. KAH03(S/P)/ 20 ng |
|
10. KAH67(X/P)/1353, 17 ng + Strep(S/P)/3200, 20 ng |
KAH134 >10:1 (Pick 2)
|
11. Strep(S/P)/ 20 ng |
|
|
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11
|
Insert DNA |
0.5 |
0.5 |
0.5 |
0.5 |
0.6 |
0.6 |
0.6 |
0.6 |
--- |
0.5 |
---
|
Vector DNA |
0.7 |
0.7 |
0.7 |
0.7 |
0.7 |
0.7 |
0.7 |
0.7 |
0.7 |
0.5 |
0.5
|
2x lgn buf (Roche) |
5.0 |
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 |
1.0
|
dH2O |
2.8 |
2.8 |
2.8 |
2.8 |
2.7 |
2.7 |
2.7 |
2.7 |
3.3 |
3.0 |
3.5
|
|
10μL |
10μL |
10μL |
10μL |
10μL |
10μL |
10μL |
10μL |
10μL |
10μL |
10μL --> R.T./ >10 min.; Add 25 μL Z-DH5α
|
|