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cAMP precipitation part II & SDS-PAGE
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Summary
Washing beads: cAMP precipitation
- Spin beads 5 min. 2100xg @ 4 °C
- Collect supernatant (= NAC)
- Add 500 μL Potter buffer
- Spin 5 min. 2100xg @ 4 °C
- Remove supernatant with vacuum pump
- Add 500 μL Potter buffer
- Spin 5 min. 2100xg @ 4 °C
- Remove supernatant with vacuum pump
- Add 500 μL Potter buffer
- Spin 5 min. 2100xg @ 4 °C
- Remove supernatant with vacuum pump and syringe
- Add 50 μL of 4x sample buffer
- Spin 5 min. 2100xg @ 4 °C
Protein work
- SDS-PAGE
- RII Overlay
- Silver / Coomassie staining
- Immunoblot
RNA work
- Preparing cDNA
- Real time qPCR
Notes
- cAMP precipitations had 50 μL of 4x loading buffer put on them, they should have been boiled for 5 - 10 min. but they were boiled approx. 2 hours
- Hardly any blue is still seen in soluble fraction, probably samples are ruined
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