Etchevers:Notebook/Genomics of hNCC/2009/04/06: Difference between revisions
(Autocreate 2009/04/06 Entry for Etchevers:Notebook/Genomics_of_hNCC) |
No edit summary |
||
| Line 6: | Line 6: | ||
| colspan="2"| | | colspan="2"| | ||
== | ==Rabbit EC, hNCC, Sigma order for Necker== | ||
1.5h endothelium + Descemet membrane from last Friday's dissection in 2.4U/mL (PBS) Dispase II cf cat 11276921-001 de Roche, thawed from freezer, old stock. 37ºC. Triturated with flame-polished Pasteur pipette. Rinsed in 20% serum/DMEM high glucose-Glutamax, then cultured in this medium in same wells. | |||
Passed R1066 cephalic and trunk cells; only counted 40K and 80K cells total, which seems very low. Instead of putting them on collagen-coated flasks (PBS, from last week in fridge), decided to reseed in 35 mm dishes in fresh Rich medium. | |||
Also took 10% of the cephalic cells (4K in theory) and seeded into one of these flasks, in fresh neural differentiation media. Shook up retinyl ester-in-DMSO, pipetted 10ul into 10 mL PBS, then added 50 ul of this to 50 mL of Stemline neural + 1x B27 (kept in fridge since last use). | |||
'''Ordered Sigma reagents:''' | |||
Biologie moléculaire = 52.34, biochimie = 52.36, culture = 52.41 | |||
* Denhardt’s solution 50x D2532 5 mL 87.00 52.34 | |||
* Cocktail inhibitor Phosphatase 1 10ul/1ml (cautharidin, bromotetramisde, mycrocystin LR in DMSO solution) | |||
Sigma, cod. P2850 P2850-1ML 50.60 recommended as 100x 52.36 | |||
* Cocktail Inhibitor Phosphatase 2 10ul/1ml (Na orthovadanate, Na m lybdate, Na tartrade, imidazide in H2O) | |||
Sigma, cod. P5726 P5726-1ML 50.60 recommended as 100x 52.36 | |||
* Pepstatin A 10ul/1ml P4265-1MG 31.20 52.36 | |||
needs to be dissolved at 1 mg/ml in 10% (v/v) acetic acid in methanol (9:1 methanol:acetic acid). Solutions of Pepstatin A can be heated as high as 60 °C without any decomposition of the peptide. Stock solutions at 1 mg/ml are stable at least a week at 4 °C. A 1 mM solution in methanol or DMSO is stable for months at -20 °C. If solutions become darker yellow, the reagent is hydrolyzing. | |||
A working concentration of 1 μM is stable for at least one day at room temperature. | |||
* Cocktail inhib. Protease 5ul/1ml (AEBSF, EDTA, aprotinin, bestatin, E-64, leupeptin in DMSO solution) | |||
Sigma, cod. P8340 P8340-1ML 38.70 52.36 | |||
* 10% sodium deoxycholate D6750-10G 13.80 52.36 | |||
* 0.1 mg/ml cycloheximide C1988-1G 99.10 or C4859-1ML 84.40 (100 mg/mL in DMSO) 52.34 | |||
* Igepal CA-630 I3021-50ML 30.20 52.34 | |||
* Triethanolamine 90278-100ML x2 bouteilles 37.10 52.34 | |||
* Stemline Neural Stem Cell Expansion Medium (urgent!!) S3194-500ML 124.50 52.41 | |||
* Laemmli Sample buffer 2x S3401-10VL 6mL 26.80 52.34 | |||
* Tween 80 P4780-100ML 14.90 52.41 | |||
* Retinyl acetate R7882-1G 29.90 52.41 | |||
1g of retinyl acetate, ref R7882 (lot 047K07091) in 3.28 mL of DMSO at 60°C = 1M (328.49 g/mol). Want 1 μM and B27 has 0.61 μM already. [http://www.hospira.com/Files/aquasol_PI.pdf Aquasol A] has 12% Tween 80, for 15 mg/mL (286.45 g/mol). = 50mM = 50,000 factor dilution to get 1 μM. | |||
--[[User:Etchevers|Heather]] 09:53, 7 April 2009 (EDT) | |||
<!-- ## Do not edit below this line unless you know what you are doing. ## --> | <!-- ## Do not edit below this line unless you know what you are doing. ## --> | ||
|} | |} | ||
Revision as of 06:53, 7 April 2009
 Genomics of human neural crest cells
|
<html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> |
Rabbit EC, hNCC, Sigma order for Necker1.5h endothelium + Descemet membrane from last Friday's dissection in 2.4U/mL (PBS) Dispase II cf cat 11276921-001 de Roche, thawed from freezer, old stock. 37ºC. Triturated with flame-polished Pasteur pipette. Rinsed in 20% serum/DMEM high glucose-Glutamax, then cultured in this medium in same wells. Passed R1066 cephalic and trunk cells; only counted 40K and 80K cells total, which seems very low. Instead of putting them on collagen-coated flasks (PBS, from last week in fridge), decided to reseed in 35 mm dishes in fresh Rich medium. Also took 10% of the cephalic cells (4K in theory) and seeded into one of these flasks, in fresh neural differentiation media. Shook up retinyl ester-in-DMSO, pipetted 10ul into 10 mL PBS, then added 50 ul of this to 50 mL of Stemline neural + 1x B27 (kept in fridge since last use). Ordered Sigma reagents: Biologie moléculaire = 52.34, biochimie = 52.36, culture = 52.41
Sigma, cod. P2850 P2850-1ML 50.60 recommended as 100x 52.36
Sigma, cod. P5726 P5726-1ML 50.60 recommended as 100x 52.36
needs to be dissolved at 1 mg/ml in 10% (v/v) acetic acid in methanol (9:1 methanol:acetic acid). Solutions of Pepstatin A can be heated as high as 60 °C without any decomposition of the peptide. Stock solutions at 1 mg/ml are stable at least a week at 4 °C. A 1 mM solution in methanol or DMSO is stable for months at -20 °C. If solutions become darker yellow, the reagent is hydrolyzing. A working concentration of 1 μM is stable for at least one day at room temperature.
Sigma, cod. P8340 P8340-1ML 38.70 52.36
1g of retinyl acetate, ref R7882 (lot 047K07091) in 3.28 mL of DMSO at 60°C = 1M (328.49 g/mol). Want 1 μM and B27 has 0.61 μM already. Aquasol A has 12% Tween 80, for 15 mg/mL (286.45 g/mol). = 50mM = 50,000 factor dilution to get 1 μM. --Heather 09:53, 7 April 2009 (EDT) | |
