RNA Prep from COS 7 cells

(Qiagen Rneasy protocol)

Pre-preparation:

Add bME to RLT (10 ul per 1ml RLT)

Add 4 vol of EtOH to RPE concentrate

Grow cells to confluency in a small

Small flask (50 ml, 25 cm²)

Confluent monolayer

Trypsinize

Wash once in PBS (pellet at 300 x g)

You can store this pellet at –70 deg if desired

Resuspend in 500 ul of Lysis Buffer (RLT)

NOTE: qiagen protocol suggests 350 ul per 5 x 10⁶ cells.

Cells per 25 cm² flask = approx _______

Homogenization:

Add cell lysate to a QIAshredder column and spin 1 min at full speed in microfuge.

Alternatively, homogenize with 5 passes through 25g needle.

Add 1 vol of Rm Temp 70% EtOH and mix by pepetting.

Apply this to an RNeasy spin column

Spin 15 sec at 8000 x g

Discard flow through

Wash the Rneasy column with 700 ul RW1, spin 15 – 30 sec at 8,000 x g.

Wash with 700 ul RPE, spin 15-30 sec at 8,000 x g.

Wash again with 500 ul RPE for 2 min!

NOTE: Be sure to spin a full 2 min to dry the membrane, since EtOH will interfere later.

Elute RNA into a fresh tube with 30 – 50 ul of RNase-free H2O, spin 1 min at 8,000 x g.

NOTE: If you expect more than 30 ug yield, elute a 2^nd time.