12/09/09
- ✓ Cultures: KAH109-116 (2 each)
- ✓ Assemblies: KAH117-125 (miR sensor Repressor/Target pairs)
- ✓ Sequencing order results
Assemblies
- KAH117: (1) KAH99/(E/S)/2913 + (4) KAH93/(E/X)/8039
- KAH118: (2) KAH100/(E/S)/3085 + "
- KAH119: (3) KAH101/(E/S)/3157 + "
- KAH120: KAH99/(E/S)/2913 + (5) KAH94/(E/X)/8211
- KAH121: KAH100/(E/S)/3085 + "
- KAH122: KAH101/(E/S)/3157 + "
- KAH123: KAH99/(E/S)/2913 + (6) KAH95/(E/X)/8283
- KAH124: KAH100/(E/S)/3085 + "
- KAH125: KAH101/(E/S)/3157 + "
> Digests (Fermentas FD)
--> KAH99, 100, 101: E/S/ApaLI
| Reagent |
1-3 |
4-6
|
30 μL/lane, 1% agarose
|
| DNA (plasmid) |
20.0 |
15.0
|
| 10x buffer |
3.0 |
3.0
|
| enzyme 1 |
1.0 |
1.0
|
| enzyme 2 |
1.0 |
1.0
|
| ApaLI |
1.0 |
---
|
| dH2O |
4.0 |
10.0
|
| |
30 μL |
30 μL --> 37°C/ ~1 hr.
|
> Measure conc.'s
| Sample |
OD260 |
260/280 |
ng/μL
|
| 1. KAH99(E/S) |
1.182 |
1.93 |
59.1
|
| 2. KAH100(E/S) |
1.003 |
1.92 |
50.1
|
| 3. KAH101(E/S) |
0.763 |
2.04 |
38.1
|
| 4. KAH93(E/X) |
0.620 |
1.84 |
31.0
|
| 5. KAH94(E/X) |
0.267 |
2.14 |
13.4
|
| 6. KAH95(E/X) |
0.206 |
1.82 |
10.3
|
> Ligations
| Ligation |
Plate results (lig : neg crtl) 12/10/09
|
| 1. KAH99(E/S)/2913, 29 ng + KAH93(E/X)/8039, 40 ng |
KAH117 1:1 (re-do)
|
| 2. KAH100(E/S)/3085, 31 ng + " |
KAH118 1:1 (re-do)
|
| 3. KAH101(E/S)/3157, 31 ng + " |
KAH119 1:1 (re-do)
|
| 4. KAH93(E/X)/ 40 ng |
|
| 5. KAH99(E/S)/2913, 28 ng + KAH94(E/X)/8211, 40 ng |
KAH120 10:1 (Pick 3)
|
| 6. KAH100(E/S)/3085, 30 ng + " |
KAH121 5:1 (Pick 3)
|
| 7. KAH101(E/S)/3157, 30 ng + " |
KAH122 2:1 (Pick 3)
|
| 8. KAH94(E/X)/ 40 ng |
|
| 9. KAH99(E/S)/2913, 28 ng + KAH95(E/X)/8283, 40 ng |
KAH123 3:1 (Pick 3)
|
| 10. KAH100(E/S)/3085, 30 ng + " |
KAH124 3:1 (Pick 3)
|
| 11. KAH101(E/S)/3157, 30 ng + " |
KAH125 3:1 (Pick 3)
|
| 12. KAH95(E/X)/ 40 ng |
|
| |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
12
|
| Insert DNA |
0.5 |
0.6 |
0.8 |
--- |
0.5 |
0.6 |
0.8 |
--- |
0.5 |
0.6 |
0.8 |
---
|
| Vector DNA |
1.3 |
1.3 |
1.3 |
1.3 |
3.0 |
3.0 |
3.0 |
3.0 |
3.9 |
3.9 |
3.9 |
3.9
|
| 2x lgn buf (Roche) |
5.0 |
5.0 |
5.0 |
5.0 |
5.0 |
5.0 |
5.0 |
5.0 |
6.0 |
6.0 |
6.0 |
6.0
|
| 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 |
1.0
|
| dH2O |
2.2 |
2.1 |
1.9 |
2.7 |
0.5 |
0.4 |
0.2 |
1.0 |
0.6 |
0.5 |
0.3 |
1.1
|
| |
10μL |
10μL |
10μL |
10μL |
10μL |
10μL |
10μL |
10μL |
12μL |
12μL |
12μL |
12μL --> R.T./10 min.; Add 25 μL Z-DH5α
|
Sequencing order results
- KAH93/MV2 - DDB122B (fwd): good
- KAH93/MV2 - DDB123B (rev): good
- KAH94/MV2 - DDB122B (fwd): good
- KAH94/MV2 - DDB123B (rev): okay (1 HS4 and one partial, then non-specific sequence)
- KAH95/MV2 - DDB122B (fwd): good
- KAH95/MV2 - DDB123B (rev): good
- KAH99/MV2 - DDB122B (fwd): good
- KAH99/MV2 - DDB123B (rev): good, polyA different from seq on file, but matches other clones
- KAH100/MV2 - DDB123B (fwd): good
- KAH100/MV2 - DDB123B (rev): good, polyA different from seq on file, but matches other clones
- KAH101/MV2 - DDB122B (fwd): good
- KAH101/MV2 - DDB123B (rev): good, polyA different from seq on file, but matches other clones
- KAH105/MV2 - DDB122B (fwd): HPK-K-Gal4DB (KAH108)? (should be Ubc-Kstop-Gal4DB)
- KAH105/MV2 - DDB123B (rev): good
- KAH106/MV2 - DDB122B (fwd): Ubc-Kstop-Gal4DB (KAH105)? (should be Ubc-K-Gal4DB); Ubc is missing a C at 21 bp
- KAH106/MV2 - DDB123B (rev): good
- KAH107/MV2 - DDB122B (fwd): Ubc-K-Gal4DB (KAH106)? (should be HPK-Kstop-Gal4DB); Ubc is missing a C at 21 bp
- KAH107/MV2 - DDB123B (rev): good
- KAH108/MV2 - DDB122B (fwd): HPK-Kstop-Gal4DB (KAH107)? (should be HPK-K-Gal4DB)
- KAH108/MV2 - DDB123B (rev):
|
Project name
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