User:Wilfred J. Poppinga/Notebook/cAMP compartmentalization/2010/06/12

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Stop stimulation: Protein and RNA/Protein isolation

Summary

All plates were washed twice with cold PBS
12 plates (6 and 6) were lysed with RIPA as described 24Feb2010 for protein isolation
2 platse (1 and 1) were lysed as described 8June2010 for RNA (and protein) isolation
- Not all of the RNA fraction could be collected without disturbing the phenol/chlororform phase, to prevent contamination less of the RNA fraction was taken
- The phenol/chlororform phase was put on ice until proteins could be collected from them
- After adding isopropanol to the RNA fraction the suspension was shaken to mix
- The RNA did not resuspend nicely in the 100% EtOH, but created white flakes
- RNA was eluted with 35 μL of RNase free water and 3 μL was taken for concentration determination
- S⁰: 507.6 ng/μL (260/280; 2.06\\260/230; 1.52)
- CSE: 610.1 ng/μL (260/280; 2.04\\260/230; 1.67)
- After precipitating the DNA with 100% EtOH the suspension was spinned down at the maximum speed 4500 RPM and no pellet was seen, this speed was to high
- The (pink) pellet obtained after isopropanol addition could not be resuspended in the guanidine HCl solution nor in the EtOH
- Only two guanidine HCl steps were performed and the EtOH incubation only for 15 min.
- Due to resuspension problems the protein pellet of S⁰ protein/RNA isolation was assisted mechanically (in the SDS solution) with a pipette tip, this suspension turned purple
- Protein pellets could not be resuspended, eventually they were left in the SDS solution @ 4 °C

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</a> </html> RNA Nanodrop