[Mike Axtell]

Thanks Phil, I really appreciate your time and advice!

I’m using the plant GITC TNA protocol 6.4. Clicking the “forum” link for protocol 6.4 leads to this forum (but I did notice the forum itself says it’s for 6.3). Sorry for confusion there.

Getting more aggressive with bead washing did help. Before I was just tumbling and flicking by hand. In my latest trial I pipetted up and down aggressively, and gave a brief vortex. I also upped the volume of 80% ethanol used to wash, and did four washes instead of two.

That did reduce the absorption at ~220-230nm, by quite a bit, but I still have not so good 260/230 ratios .. around 1.0 to 1.2. Nucleic acid yield is about what I expect given the tissue inputs I am using. Just wish I could get it even cleaner. I’ll keep running down some other variables and post again if I manage to fully solve it. And yes if your NZ colleagues had any ideas I would welcome them.

Some googling led me to fact that Iron(III) and maybe other Iron compounds also absorb down there in the 220-230nm range. I wonder if residual nanoparticles or free Iron is it?

Thanks again!

Mike

 

 

[Mike Axtell]

Thanks Phil, I appreciate your advice. I tried a series of extractions holding tissue amount constant (~ 18mg Arabidopsis leaves) and increasing silica bead usage over a ten-fold range. More beads indeed reduced clumping. However, the strong GITC contamination in the eluted TNA was not affected at all by the bead amounts.

I also did the same experiment (varying bead amounts) in mock extractions, with no plant tissue at all, with the same result: Very strong levels of GITC (inferred from absorbance 220-230nm) in all conditions, in the absence of any biological input.

I’m following the 6.4 protocol quite closely, and I remade all solutions in this most recent trial. The only small difference is that I use “LTE-7.5” to elute the TNA, instead of water (LTE-7.5 is 10mM Tris-HCl pH 7.5, 0.1mM EDTA). The silica-coated beads I am using were synthesized according to the BOMB protocol, and everything seemed as expected : good yield, no evidence of oxidation upon storage.

Any other suggestions?

Thanks!,

Mike Axtell

[Mike Axtell]

Hi,

I’m seeing strong absorbance at ~ 220-230 nm with TNA from this protocol (6.4). Suspect it is carryover of GITC. Also noticing that my silica beads tend to clump a little during binding, and only seem to “de-clump” upon elution. Wondering if the two observations (Guanidinium carryover and bead clumping) are connected, but even if unconnected, what I’d really like to solve is the carry-over of GITC into the final TNA eluate.

 

Thanks!

 

[Mike Axtell]

Has anyone examined the recovery of microRNAs (~21 nt RNAs) using this TNA protocol, or with any of the BOMB protocols?

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