[PStepper]

Hi everyone,

this is the main discussion space for BOMB protocol #8.1 RNA extraction from mammalian cells using TRI reagent.
Here you can post all your questions and feedback!

[PStepper]

reserved space

[Tonya]

Hello!

If I were to adopt this protocol to further clean up my RNA from TRIzol and chloroform contaminants, could I just simply add the RNA binding buffer + beads and proceed from step 12? And do you know what the minimal volume that has been used to elute the RNA?

(If this addressed elsewhere, I apologize! Would you direct me to the right location?)

Thanks!

[Tomek]

Dear Tonya,

With the protocol you can get the RNA from TRIZOL solubilised samples directly without adding chloroform. Yet, if you added chloroform already and have separated the water and phenolic phases, you could add the RNA binding buffer to the water phase (where the RNA is), with 4 times the volume of the binding buffer to 1 volume of the sample (indeed, like from the step 12 🙂 ).

The minimal volume will depend on the amount of beads that you use. As a rule of thumb, these should be covered with the liquid completely, and of course some of the liquid will be stuck in between the beads. What we typically use for RNA extraction is around 2 ul of the 1:1 beads:water stock. Meaning, one could go quite low with the elution volume, yet one has to take into consideration that not all the liquid you add, you will be able to take out. Alternatively, one can elute in bigger volume and speed-vac to concentrate.

Cheers and hope this helps,

Tomek

[admin]

all replies concerning adaptation of this protocol for SARS-CoV-2 RNA extraction have been moved to

Adjusting BOMB protocol #8.1 for SARS-CoV-2 extraction

 

[Volker]

Dear BOMB community,

first of all, let me say that I am a big fan of your work! We have just recently used the 3D models to print some magnetic racks and they work really well.

Now concerning the selfmade Tri reagent, also that seemed to work great. I just tested that reagent side-by-side with commercial reagents and yield as well as 260/280 ratios were almost identical.

My question aims at the Tri reagent itself: does anybody have any experience with using (organic) dyes to further color the organic phase (as in the original Trizol)? I know that the 8-hydroquinoline already gives this nice yellow color, but I would like to explore further options. Out of curiosity, I have tried Acridin Orange (mostly in organic phase, but aqueous phase looks somehow cloudy), Xylenecyanol and Direct Red (both dyes are in both phases). I will test some water-insoluble solvent dyes in the near future, but maybe somebody else already figured out which dyes work well.

Any feedback would be appreciated. Once again, thank you guys for the really great protocols and resources!

Cheers,

Volker

[admin]

Ahoy,

Welcome and thanks for the flowers.

Unfortunately I cannot give you a clear answer. This seems to bee a rather common question on the interweb, sadly with little to no answers. I would suggest trying Fuchsin (based on this article https://jb.asm.org/content/jb/52/1/99.full.pdf).

Colour wise it is strikingly close to the one in probrietary Trizol. As it is frequently used in histology it should not be too hard to come by.

Cheers

Tim M

 

[Volker]

Thank you Tim for the quick reply!

I will check out Fuchsin as well and give feedback once I find something useful for the community.

Cheers,

Volker

[admin]

Awesome, please do.

[Author]

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