TITLE: Zymo Duet DNA/RNA MiniPrep Plus Extractions
AUTHORS: Amy Lyden

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Attached are detailed instructions for following the kit guidelines for the Zymo Duet DNA/RNA MiniPrep Plus.</div></div>

[STEPS]
?. [Sample Preparation for cultured isolates]
Add 1X DNA/RNA shield (2x concentrate diluted in nuclease-free water) to bead bashing tubesTissuelyse on Qiagen Tissuelyser II   at a frequency of 1/30 s. Alternatively, vortex at the highest frequency for 2 minutes.Spin  at 14000RCF.Add of DNA/RNA Lysis buffer. Proceed with Nucleic Acid Purification.
320 µl
[minutes]
320 µl
?. [DNA Purification]
DNA is bound to the yellow column. Transfer the Spin-Away™ Filter (yellow) into a new Collection Tube. Proceed with Nucleic Acid Purification.
?. [RNA Purification]
Transfer flow-through into Eppendorf if necessary. Add an equal volume of ethanol (95-100%) to the flow-through and mix well.
?. [Nucleic Acid Purification]
Add  DNA/RNA Prep Buffer to the (yellow/green) column and centrifuge at 16000 RCF. Discard the flow-through.
400 µl
?. [Nucleic Acid Purification]
Transfer up to of the sample into a Spin-Away™ Filter (yellow) in a Collection Tube and centrifuge  at 16000 RCF. If you have > , you can reload the column and centrifuge again. Save the flow-through if you wish to proceed with RNA Purification. If not, discard flow through, and skip RNA Purification steps. (For whole blood ONLY, after doing the above, discard flow-through. Add of DNA/RNA Lysis buffer directly to the filter matrix. Let stand for . Then centrifuge  at 16000 RCF. SAVE this flow through as your RNA is in it)
700 µl
[seconds]
700 µl
200 µl
[minutes]
[seconds]
?. [RNA Purification]
Transfer up to of the sample with ethanol into a Zymo-Spin™ IIICG Column (green) in a Collection Tube and centrifuge at 16000 RCF. Discard the flowthrough. Repeat by reloading the column if you have more sample remaining.
700 µl
[seconds]
?. [Nucleic Acid Purification]
?. [RNA Purification]
Wash the column with  DNA/RNA Wash Buffer and centrifuge at 16000 RCF. Discard the flowthrough.
400 µl
[seconds]
?. [RNA Purification]
Add 80 µl DNase I Reaction Mix (reconstituted DNAse + DNA Digestion Buffer) directly to the column matrix. (can make a master mix of the same proportions for multiple samples)
5 µl
75 µl
?. [RNA Purification]
?. [RNA Purification]
Incubate the column at room temperature (-) for . Proceed with Nucleic Acid Purification.
[minutes]
?. [Nucleic Acid Purification]
Add DNA/RNA Prep Buffer to your (yellow/green) column and centrifuge at 16000RCF. Discard the flow-through.
400 µl
[seconds]
?. [Nucleic Acid Purification]
Add  DNA/RNA Wash Buffer and centrifuge at 16000RCF. Discard the flow-through.
700 µl
[seconds]
?. [Nucleic Acid Purification]
Add DNA/RNA Wash Buffer and centrifuge the column for at 16000RCF to ensure complete removal of the wash buffer. Carefully transfer the column into clean microcentrifuge/Eppendorf tubes.
400 µl
[minutes]
?. [Nucleic Acid Purification]
Add  DNase/RNase-Free Water directly to the column matrix, let stand for , and then centrifuge  at 16000RCF to elute DNA and RNA from the respective column.
50 µl
[minutes]
[seconds]
?. [Nucleic Acid Purification]
Take all  of eluted DNA/RNA in microcentrifuge tube and reload onto column. Let stand for , and then centrifuge at 16000RCF to elute more DNA and RNA from the respective column.
50 µl
[minutes]
[minute]
?. [Nucleic Acid Purification]
Aliquot RNA into single use aliquot tubes and freeze at -80. Freeze DNA at -20 or -80.
?. [Sample Preparation for liquids (ie, plasma, tracheal aspirate, CSF)]
Add 1 part sample to 3 parts DNA/RNA lysis buffer (ie, sample to lysis buffer) in a bead bashing tubeTissuelyse on Qiagen Tissuelyser II   at a frequency of 1/30 s. Alternatively, vortex at the highest frequency for 1 minutes.Spin  at 14000RCF. Proceed with Nucleic Acid Purification.
100 µl
300 µl
[minutes]
?. [Nucleic Acid Purification]
Label a Spin-Away Filter (yellow) and the microcentrifuge tube for each sample.
?. [Sample Preparation for samples in DNA/RNA shield (1x)]
Add one part DNA/RNA Lysis buffer (ie of sample in DNA/RNA shield to of lysis buffer). Proceed with Nucleic Acid Purification.
200 µl
200 µl
?. [Sample Preparation for whole blood]
Add of 2X DNA/RNA shield to each  of b2 of blood sample and mix throughly.For every of reagent/blood mixture, add of reconstituted Proteinase K and mix thoroughly. Incubate at at room temperature for
200 µl
200 µl
400 µl
8 µl
[minutes]
?. [Nucleic Acid Purification]
Proceed using the yellow column with DNA Purification sectio,  and using the flow-through with the RNA purification section. After that, you will continue with the Nucleic Acid Purification for both DNA and RNA in parallel.