DNA extraction from Filters with UltraClean Soil kit. - (Nov/11/2008 )
Anyone here have experience with Mo Bio Lab's soil kit?
I'm trying to use the UltraClean Mega Soil kit to extract DNA off of filters that rumenal fluid has passed through.
I would like to do a set with DNase treatment and one without, because I'm concerned about free DNA from ingested plants and cow cell DNA.
For instance for the DNase treated set - I would like to:
- soak the filters in TM buffer inside a 50 ml tube
- add 40 units DNase and incubate at 37C for one hr
- deactivate the DNase with 100mM EDTA and 100mM EGTA
- add the buffer+filter together to to the bead tube.
- proceed with vortexing and the remainder of the protocol provided by mo bio
For the sample without DNase, could I just add the filter and the bead solution to the bead tube, vortex for 10 min, freeze/thaw - then proceed as normal with solution S1?
I appreciate any advice
You could do a DNase digest to remove free contaminating DNA as you planned but why would you do it in 100mM EDTA/EGTA? That would inhibit the DNase. Also I would suggest you try either AquaGenomic or AquaRNA for your DNA extraction as well. Simply add the filter to AquaGenomic or AquaRNA solution, beat it up, spin to pellet debris, then precipitate the DNA from the supernatant with isopropanol or AquaPrecipi, if your sample contains tons of PCR inhibitors.
Hi yes I am aware of the inhibition, I am adding the EGTA/EDTA AFTER the digestion. I am lysing the cells for analysis afterwards so I need to deactivate the enzyme.
Thanks for the advice.