Spin Column-Based Extraction
PROTOCOL DUMP PLEASE FORMAT
Things to check before starting the protocol
Make 1 mg/mL lysozyme in RNase-free 1x TE buffer [prepare 150 µL per RNA sample]
Add 10 µL β-Mercaptoethanol (βME) to 1 mL Lysis Solution (Norgen Kit) in an RNase-free eppendorf tube and mix by quick vortexing [prepare 400 µL per RNA sample required]
Thaw an aliquot of yeast tRNA (25 µg/µL stock concentration) on ice
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 labwork, e.g. minipreps.
Protocol Steps
Spin down at room temperature 5 mL of bacterial culture at OD600 0.2 (log phase) at max rpm (4,750 rcf), and discard the supernatant [try to withdraw as much supernatant as possible without disturbing the bacterial pellet]
Resuspend the pellet in 100 µL of the lysozyme solution (in TE) by pipetting up and down and/or vortexing and transfer to an RNase-free eppendorf tube. Incubate the resuspension at room temperature for 5 minutes [if the room temperature is low, incubate at 25 °C]
Add 300 µL of the βME containing Lysis Solution (Norgen Kit) and mix by vigorous vortexing for 10-15 seconds
Add 200 µL of ethanol (98-100%) to the lysate and mix by vigorous vortexing for 10-15 seconds [use the EtOH bottle from the designated "RNA ONLY" drawer]
Assemble the RNA purification column and the collection tube (Norgen Kit) and add 600 µL of the lysate from Step 4 above to it. Spin at 14,000 rcf for 1 min at room temperature [if all the lysate does not go through the column, spin for another 1 min]
Discard the flow-through and reassemble the column
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]
Discard the flow-through and reassemble the column
Repeat Steps 7 & 8 two more times (total of 3 washes)
Spin the column at 14,000 rcf for 2 min at room temperature in order to dry the column resin
Discard the collection tube and place the column into a fresh RNase-free eppendorf tube [use cold eppendorf tubes]
Add 50 µL of the Elution Solution (Norgen Kit) to the column and spin at room temperature at 200 rcf for 2 min, followed by 14,000 rcf for 1 min [place the RNA on ice-water immediately and keep cold all the time]
For greater recovery, place the column back into the same eppendorf tube and repeat the elution with another 50 µL of the Elution Solution
Take out a 3 µL aliquot in a separate RNase-free eppendorf tube for later analysis
To the rest of the 95 µL RNA, add 9.5 µL of the 10x TURBO DNase Buffer and 2 µL TURBO DNase (TURBO DNA-free™ Kit) and mix gently by flicking the tube
Incubate at 37 °C for 30 min
Add 10.7 µL DNase Inactivation Reagent (TURBO DNA-free™ Kit) and mix gently by flicking the tube [before pipetting the DNase Inactivation Reagent, flick the tube a couple of times to ensure that the beads are uniformly dispersed]
Incubate at room temperature for 5-6 min while mixing gently by flicking the tube every 2 min
Spin at room temperature at 10,000 rcf for 1.5 min and transfer 90 µL of the supernatant to a fresh RNAse-free eppendorf tube [use cold eppendorf tubes; keep cold all the time]
Take out a 3 µL aliquot in a separate RNase-free eppendorf tube for later analysis [use cold eppendorf tubes; keep cold all the time]
Use the nanodrop to determine RNA concentration using the aliquots taken earlier in Steps 14 and 20 (pre- and post- DNAse treatment)
Using the Elution Solution (Norgen Kit) dilute all the post-DNase RNA samples to the same concentration 50-150 ng/ µL
Run 1 µL each of the RNA samples from Steps 14 & 20 above (pre- and post- DNAse treatment) on a 1% agarose gel to check for RNA integrity.
-Two sharp bands at ~1 kb confirm RNA integrity;
fuzzy/ smeary bands indicate degradation
Aliquot 20 µL each of the RNA samples from Step 22 into four fresh RNase-free tubes a-d
Add 0.2 µL of yeast tRNA (25 µg/µL stock concentration) to two aliquots (a & b) from Step 24 above [end concentration 250 ng/µL tRNA]
Freeze down all the five tubes at -80 °C: the original tube from Step 22 and tubes a-d from Steps 24 & 25.
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