#1    RNA Isolation – QUICK AND EASY

SurePrep TrueTotal RNA Purification Kit – Fisher BP2800-50

Notes: hands, saliva, pipettes can have a lot of RNAses. Important to
wear gloves, and clean gloves and pipettes with RNAse away spray
solution for decontamination


1.      Grow cells in a ~60-100% full 60-mm dish or 6-well plate ~1-3x10^6
cells  Lift cells by trypsin, spin, aspirate media, save pellet at
–80C until use

2.      Lyse cells:
a.      Lysis solution: optional- add 10ul 2-BME to each ml of solution
(helps decrease RNAse activity)
b.      Add 350ul lysis solution directly to frozen cell pellet
c.      Pass lysate 3x through a 25-26 gauge syringe/needle to shear genomic DNA
d.      Spin lysate microcentrifuge RT 1min max speed to pellet debris. Use
supernatant (clarified lysate) for next step.

3.      Bind RNA to column
a.      Assemble column with one of the provided collection tubes
b.      Add 200ul 95-100% EtOH to clarified lysate, vortex 10 seconds
c.      Place lysate/EtOH mixture in the column, Spin 1min RT 12,000rpm
d.      Discard flowthrough

4.      Column Wash
a.      Apply 400ul Wash Solution to the column, spin 1min RT 12,000rpm. If
entire volume of wash solution has not passed through the column into
the collection tube, repeat spin step.
b.      Discard flowthrough
c.      Repeat steps a-b to Wash a second time
d.      Wash column a third time with another 400ul Wash solution and this
time, spin 2 minutes
e.      Ensure the column is dry by spinning an additional 1min
f.      Discard collection tube and flowthrough

5.      RNA Elution
a.      Place the column into a fresh 1.7ml elution tube provided with the kit
b.      Add 50ul Elution Buffer to the column (center of membrane)
c.      Spin 2min 1,500 rpm followed by 1min 12000 rpm.
d.      Put the 50ul back in the column and re-spin for 1min 12000rpm to
enhance RNA yield obtained
e.      Look at the column and if entire 50ul has not been eluted, repeat
1min 12000rpm spin.

6.      Measure RNA concentration:
a.      4ul RNA + 96 TE (1:25 dilution)
b.      You should have hopefully ~1-2 ug/ul RNA
A260 x dilution factor x 40 (conversion factor for 1ug) = ug RNA/mL
c.      Freeze at –80C until cDNA synthesis experiment