So I'm using TRIzol for RNA extraction, but suddenly I'm getting no pellet during the isopropanol step *even though I added glycogen.*

A few weeks ago I:

1. Took a large quantity of bacteria

2. Resuspended in TRIzol

3. Made a bunch of aliquots

4. Stored them all at -80

And those couple weeks ago, I was getting~50 ug of RNA per aliquot (with good RINs, and they looked great on RNA gels).

Yesterday, I took another aliquot and tried to prep it using the exact same process, and got nothing. No pellet appeared during the isopropanol precipitation step even though I added GlycoBlue.

I continued the purification to see if the pellet was just hard to see: ~0 ng/uL by nanodrop

Did I just make a pipetting error? Was my isopropanol bad? No: I repeated the repeated the process *again* with completely new sample, completely new isopropanol, and still no pellet at all.

I'm confident I'm lysing my samples well. I optimized the lysis a while ago, but even before optimization, my yields were never this bad.

I'm sure I added the GlycoBlue. I watched it diffuse into my sample.

I'm sure I added isopropanol. I watched the alcohol/water mix and used brand new isopropanol.

I'm sure I mixed the isopropanol/aqueous phase. I watched it carefully.

I'm sure I actually spun my samples. I tried it over and over.

Protocol:

1. Take bacteria+TRIzol aliquot from -80 (which used to give ~50 ug)

2. Lyse via bead beating (same beads, same bead beater, same settings, same duration that gave 50 ug in the past)

3. Add 0.2 V chloroform

4. Centrifuge 12k xg for 15 min

5. Take upper, colorless aqueous phase

6. Add 1 V of RT isopropanol

7. Add ~30 ug GlycoBlue

8. Invert a bunch of times to mix well

9. Incubate 10 min RT

10. Centrifuge ~20k xg for 10 min

Nothing. No pellet at all. Even with glycogen.

I tried spinning again: No pellet

How is this possible?