User:Karmella Haynes/Notebook/Polycomb project/2010/05/06

From OpenWetWare
Jump to: navigation, search
Owwnotebook icon.png Project name Report.pngMain project page
Resultset previous.pngPrevious entry      Next entryResultset next.png

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
File:KAH 050610 gel1.tif
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:

  1. KAH126-1 (3/14/10)
  2. KAH127-4 (3/09/10)
  3. KAH128-8 (3/09/10)
  4. KAH129-4 (3/14/10)
  5. KAH130-2 (3/09/10)
  6. KAH132-8 (3/09/10)
  7. 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.