T7 RNAP Reaction Clean-Up

PROTOCOL DUMP PLEASE FORMAT

The RNA produced from a T7_in vitro_transcription reaction can be purified using Norgen spin columns, or byphenol:chloroform extraction.

Number of samples:

1-3 x #T7_in vitro_transcription reactions

• Note: this depends on your expected yield, and how much RNA you need. T7 reactions using NEB's HiScribe kit can yield up to 180 ng/uL, and the spin columns used in this protocol can capture 50 ug of RNA.

To prepare beforehand:

1. 6 microcentrifuge tubes per sample (label

   a

   thru

   f

   ). Put on ice.

2. 300 uL Buffer RL per sample. Add 3 uL β-mercaptoethanol per every 300 mL Buffer RL

3. 300 uL isopropyl alcohol per sample

4. 800 mL 1X TAE

5. 1. Add 16 mL 50X TAE and ddH

      2

      O to 800 mL

6. Aliquot out DEPC water from container - 150 uL per sample

Steps to minimize RNAse contamination

• Cover hands, arms, head, and body with sterile protective gear (RNAses are in your saliva, your tears, and on your body surface).

• Wipe down all bench surfaces with RNAse Away

• Use only sterile, disposable plastic tubes, tips, and serological pipettes. It is important to keep a box set of tips for RNA use only -- do not use these tips for any other lab work, e.g. minipreps.

Protocol Steps

Sample preparation

1. Split each T7 reaction into the desired number of samples in tubes (a). Add DEPC H

   2

   O to 100 uL to each sample.

2. Add 250 uL Buffer RL to sample. Vortex 10 seconds

3. Add 250 uL isopropanol. Vortex 10 seconds.

Column binding and washing

1. Assemble the RNA purification column and the collection tube (Norgen Kit) and add the 600 µL of prepared sample. Spin at 3,500 rcf for 1 min at room temperature (

   if all sample doesn't go through, spin 1 more min at 14,000 rcf

   )

2. Discard the flow-through and reassemble the column

3. Add 400 µL of the Wash Solution (Norgen Kit) to the column and spin at 14,000 rcf for 1 min at room temperature [the Wash Solution should have been prepared beforehand by adding RNase-free ethanol to the Norgen supplied bottle]

4. Discard the flow-through and reassemble the column

5. Repeat wash 2 more times (total of 3 washes)

6. Spin the column at 14,000 rcf for 2 min at room temperature in order to dry the column resin.

Elution

1. Discard the collection tube and place the column into tube (

   b

   )

2. Add 50 µL of the Elution Solution (Norgen Kit) to the column. Let stand 2 minutes.

3. Centrifuge 2 min at 200 rcf, followed by 2 minutes at 5,800 rcf. Spin at 14,000 rcf for 30 s.

4. For greater recovery, place the column into a new eppendorf tube (

   c

   ). Add the previous eluate to the column and let stand 2 minutes at RT. Centrifuge 1 min at 200 rcf, followed by 2 minutes at 5,800 rcf. Spin column at 14,000 rcf for 30 seconds.

TURBO DNasereaction

1. To tube

   (c)

   , add 4.5 uL TURBO DNase buffer and 2 uL TURBO DNase, and mix gently via repipetting.

2. Incubate at 37C for 30 minutes.

3. Resuspend DNase Inactivation Buffer by flicking the tube. Add 10.3 uL of this to each sample.

4. Incubate 6 min at RT, flicking to mix every 2 min.

5. Centrifuge at 10,000g for 1.5 minutes to pellet DNase Inactivation Buffer.

6. Transfer 45 uL RNA-containing supernatant to its final tube (

   d

   )

Measuring concentration and purity

Note: start to prepare gel before this step.

1. Transfer 1 uL of RNA solution to 9 uL DEPC H2O in tube

   (e)

   .

2. Transfer 2 uL of this solution to a separate microcentrifuge tube (

   f

   ).

3. Measure concentration in tube (

   f)

   using Nanodrop.

4. Run tube

   (e)

   on non-denaturing TAE gel.

Native TAE-agarose gel

1. Thoroughly clean large gel box and small combs with RNase Away.

2. Measure out 1.5 g of SeaKem agarose. Add to autoclaved small (500 mL) flask. Add 150 mL 1X TAE. Microwave for ~60 sec, or until agarose is dissolved.

3. Wait for molten gel to cool to 60C. Add 3 uL GelStar and swirl to mix. Pour into gel caster, and put 2 small combs. Let stand until solidified, about 15 min.

4. To tube (

   e)

   , add 8 uL Formaldehyde RNA Loading Dye and pipet to mix.

5. To prepare ssRNA ladder: add 10 uL Formaldehyde RNA Loading Dye to 10 uL ssRNA ladder.

6. Set up and load gel

   . Run at 80 V. You should see significant band migration in the gel after around 30 min.

7. HOW TO TELL IF RNA IS DEGRADED: A LOW molecular weight smear (below the main band) and/or an extra-fuzzy band indicate that the RNA has degraded.