For whole nuclear protein extraction, nuclei were homogenized in urea extraction buffer (10 mM Tris pH 8, 100 mM NaH2PO4, 8 M urea) for 10 minutes at room temperature (RT), spun at 16.000 g for 10 minutes at RT and analyzed by Western blot.
At RBM, the samples were thawed at room temperature (RT), vortexed, spun at 13,000g for 5 minutes for clarification, and 1.0 mL was removed into a master microtiter plate for MAP analysis.
Spin around, spin, spin, spin.
Red blood cells were lysed with 2 ml 1 × FACS Lysing Solution (BDIS; 10 min, RT), samples were spun down (500 g, 5 min) and the cell pellet resuspended in 200 μl CellFix (BDIS) for analysis.
Let's see how they spin this RT @BarackObama The new healthcare plan includes mandatory death sentences for anyone that spoils Breaking Bad.
Spin at RT at 3,000 rpm to pellet cells.
After a spin down, 0.4 mM dNTPs, RT buffer 1X, 200U M-MLV RT (Promega) and 25U RNAsin (Promega) were added, in a total volume of 25 µl and incubated 1 h at 42°C.
After DNA digestion, RNA was either purified by standard procedures using a mixture of phenol/chloroform/isoamyl alcohol and chloroform/isoamyl alcohol (for RT-PCR) or RNeasy® MinElute™ spin columns (Qiagen) (for microarray and RNAseq).
RT-PCR products were subsequently purified using spin prep columns (PeqLab), ligated into the pGEM T-Easy vector (Promega) and transformed into DH5α or TOP10F' bacterial cells.
