05/06/10
- ✓ Minipreps: FlpE (4)
- ✓ X-linked chromatin prep & sonication: Dox-induced samples from 3/09/10 and 3/14/10; practice run for U2OS plain only (with Qingqing)
- ✓ FlpE: cultures (4); streak 100μg/mL plate with clone #3
Minipreps
> Check with E, P, and E/P digests
| Reagent |
1,4,7,10 |
2,5,8,11 |
3,6,9,12
|
Expected:
1. FlpE-1, E = 7160, 546
2. FlpE-1, P = 7706
3. FlpE-1, E/P = 5649, 1511, 546
4. FlpE-2, E = 7160, 546
5. FlpE-2, P = 7706
6. FlpE-2, E/P = 5649, 1511, 546
7. FlpE-3, E = 7160, 546
8. FlpE-3, P = 7706
9. FlpE-3, E/P = 5649, 1511, 546
10. FlpE-4, E = 7160, 546
11. FlpE-4, P = 7706
12. FlpE-4, E/P = 5649, 1511, 546
|
15 μL/lane; 1% agarose
|
| DNA(plasmid) |
2.0 |
2.0 |
2.0
|
| 10X buffer |
1.5 |
1.5 |
1.5
|
| EcoRI |
1.0 |
--- |
1.0
|
| PstI |
--- |
1.0 |
1.0
|
| dH2O |
10.5 |
10.5 |
9.5
|
|
|
15 μL --> 37°C/ ~15 min.
--> Conclusion: Success! Combine 4 preps (300μL total) for DNA clean-up (tomorrow)
--> Streak clone #3 (saved toothpick) onto 100 μg/mL Amp plate.
--> Inoculate 4 5 mL cultures and 1 2 mL culture (for glycerol stock) w/ 2 μL left-over culture #3.
X-linked chromatin prep & sonication
> Full sample set is numbered as follows:
- KAH126-1 (3/14/10)
- KAH127-4 (3/09/10)
- KAH128-8 (3/09/10)
- KAH129-4 (3/14/10)
- KAH130-2 (3/09/10)
- KAH132-8 (3/09/10)
- U2OS plain (3/14/10)
> Today, do chromatin prep and sonication for U2OS plain only ...Oops, grabbed #6 instead of #7 (5/21/10)
> Completion time: ~2 hours for chromatin prep, ~2 hours per sample for sonication
> No more than two samples should be done per day, since sonication must be done one sample at a time.
> Finish at DNA quality control step. Pronase A treatment of 100 μL sample (overnight). Flash-freeze remaining ~6 mL and store at -80°C.
|
Project name
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