User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/07/01: Difference between revisions
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*Solaris qPCR Gene Expression Master Mix (2x) (ROX) | *Solaris qPCR Gene Expression Master Mix (2x) (ROX) | ||
*qPCR cycler (ABI StepOne) | *qPCR cycler (ABI StepOne) | ||
*MicroAmp™ Fast optical 48-well reaction plate (0.1 mL) (Applied Biosystems) | |||
=====Primer===== | =====Primer===== | ||
*Primer AKAP9 ([http://www.dharmacon.com/CatalogSearch/ConsolidatedSearch.aspx?searchTerm=AKAP450&searchTarget=10142_+AKAP9 Dharmacon Inc.] | *Primer AKAP9 ([http://www.dharmacon.com/CatalogSearch/ConsolidatedSearch.aspx?searchTerm=AKAP450&searchTarget=10142_+AKAP9 Dharmacon Inc.] |
Revision as of 09:36, 2 July 2010
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SummaryMaterials & MethodsMethodBradford
SDS-PAGE
Coomassie staining
Silver staining
1. Fix gel for 2 h in fixing solution (30% ethanol (v/v) & 10% acetic acid (v/v)) 2. Wash gel 2x 10 min. with 250 mL 10% 30 Ethanol (v/v) 3. Wash gel 3x 10 min. with dH2O 10 min. OPTIONAL a. Soak for 10 min in 3.4 mM potassium dichromate-0.0032 N nitric acid (200 ml/gel, 25°C) (stronger signal) b. Wash with deionized water three times for 10 min (400 ml/gel, 25°C). STAINING 4. Incubate gel 30 min. in 200 mL 0.1% Silvernitrate solution (w/v) 5. Rinse 3x 5 min. with dH2O DEVELOPING 6. Develop gel for 5 – 10 min. in 200 mL 3% NaCO3 + 200 uL 37% Formaldehyde solution 7. Stop development with 200 mL 1~5% Acetic acid for 5~10 min. 8. Wash gel with dH2O (2 min.) DESTAINING 9. Destain with (Farmer’s reducer, will clear whole gel!) a. 50 mL 2% Potassium hexacyanoferrate(III) (C6N6FeK3) b. 50 mL 3% sodiumthiosulphate (Na2S2O3) 10. Wash 3x 10 min. with dH2O (RESTAINING) (Drying the gel, gel can be soaked in drying solution (10% glycerol + 15% EtOH) for 30 min. and let to dry qPCRPreparing cDNA
The following procedure is designed to convert 1 pg to 5 μg of total RNA or 1 pg to 500 ng of poly(A)+ RNA into first-strand cDNA:
Real time qPCR
Primer
Notes
Results
Discussion
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Attachment<html> <a href="http://openwetware.org/wiki/Image:Bradford_01072010.xls"> <img src="http://openwetware.org/images/f/f8/Owwnotebook_icon.png" alt="" width="50" height="50"> </a> </html> Bradford results, slightly different than those calculated by Tina <html> <a href="http://openwetware.org/wiki/Image:HTERT_Donor_12_ht31_160310.xls"> <img src="http://openwetware.org/images/f/f8/Owwnotebook_icon.png" alt="" width="50" height="50"> </a> </html> Excel TITLE |