Preparing an RNAse-free setup

This is a protocol for preparing an RNAse-free setup.

Materials needed:

• RNAse Erase
• RNAse free pipette tips, centrifuge tubes, and water
• 70% EtOH (freshly opened, to ensure it is RNAse free)

For instance, the following were ordered from VWR: 1 bottle RNAse Erase (IC821682), 1 pack RNAse free water (101419-818), 1 pack of 960 1-200uL tips (53509-815), 1 pack of 1000 0.65mL tubes (87003-290), and  pack of 500 1.7mL tubes (87003-294).

Equipment needed:

• Barnstead water ultrapurification system

A. A few overarching cautions:

• Spray your gloves periodically with RNAse Erase and 70% EtOH.
• NEVER reach over your RNAse-free workspace, or breathe or cough onto your setup. These actions could easily contaminate your RNA with RNAse’s.

B. General procedure for getting rid of RNAse

1. Rinse three times with RNAse Erase (spray RNAse Erase into its cap for easy use)

2. Rinse three times with 70% EtOH (pour some into the RNAse Erase cap for easy use)

3. Rinse three times with DI H₂O (straight from Barnstead, into the RNAse Erase cap)

C. Pipette tips

1. Transfer rack of pipette tips to a RNAse-free tip box.

2. Autoclave on setting #4.

3. While working with the tips, keep the box top closed at all times so the tips stay RNAse free.

D. Centrifuge Tubes

1. Obtain a beaker that has been autoclaved. Treat with general procedure to get rid of RNAse.

Carefully open the box of centrifuge tubes. Open the protective plastic bag without touching the inside of the bag.

2. Using tweezers that have been thoroughly sprayed with RNAse Erase, carefully remove centrifuge tubes one at a time, place into cleaned beaker and autoclave.

E. Micropipetters

1. Perform the three rinses described in A. directly into the micropipetter

F. Workbench

1. Organize what you will be using on your bench (i.e. RNAse Erase, pipette tips, centrifuge tubes, water, bucket of ice with samples). Everything should be in easy reach and in a location where you will not have to reach over your RNA experiment.

2. Spray the bench (and your gloves) with RNAse Erase. Wipe bench dry with a Kimwipe.

3. Spray the bench (and your gloves) with RNAse-free 70% EtOH. Wipe bench dry with a Kimwipe.

4. Two-three Kimwipes into the box, the Kipwipes are considered to be very sterile (and RNAse-free). Pull out two Kimwipes, then take the next two Kimwipes out and lay them on your bench as an RNAse-free workspace (can place tubes, etc. here).