Dec 11, 2024
Zymo DNA/RNA MiniPrep (milton extractions)
This protocol is a draft, published without a DOI.
- Maggi Mars1
- 1usf
Protocol Citation: Maggi Mars 2024. Zymo DNA/RNA MiniPrep (milton extractions). protocols.io https://protocols.io/view/zymo-dna-rna-miniprep-milton-extractions-drk554y6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: November 12, 2024
Last Modified: December 11, 2024
Protocol Integer ID: 111997
Keywords: zymo dna, seawater filter, rna miniprep, zymo, dna, rna, filter
Abstract
Zymo extractions from seawater filters
Troubleshooting
Preparation
15m
Wipe bench and hood with RNAse ZAP (this is a detergent and it soapy)
Wipe bench and hood with RNAse away (base)
Wipe bench and hood with 70% Ethanol
Turn on the UV light in the hood (15 minutes)
15m
Thaw samples at room temperature
Prepare DNase-1 in digestion buffer - DNAse aliquots for 10 samples are in the -20 freezer drawer in a 50 ml falcon tube - allow to thaw in the fridge while cleaning the bench or at room temp if you are ready to use it immediately upon thawing. Make enough for 10 samples, even if you are doing less - transfer 50µl aliquot from 0.5 ml tube to a full-size 1.5 ml eppendorf tube. Add 750µl of DNA Digestion Buffer (from kit) and mix gently by pipetting up and down. Keep in the fridge until you are ready to use.
RNA + DNA extraction
43m
Arrange samples symmetrically on vortex tube adapter - shake on high for 15 minutes (page 5, step 2)
15m
While shaking samples, set up and label the amount of Eppendorf tubes and filter columns (YELLOW and GREEN only) needed throughout the extraction
Centrifuge all bead tubes at 16,000x G for 30 seconds 16000 rpm, 00:00:30
30s
Don't forget to make note of which sample corresponds to which number written on the tubes
Use the 1000 µl pipette (blue) set to 1000, remove as much liquid as possible - start by removing all liquid above the beads and filter. Then use the pipette to press the filter against the side of the tube and remove any additional liquid above the beads. Finally, put the pipette tip down into the beads and remove the last bit of liquid. All liquid is moved over into a fresh 1.5 ml Eppendorf tube.
Centrifuge eppendorf tube with sample at 16,000x G for 30 seconds 16000 rpm, 00:00:30
30s
remove 400 µl of supernatant (avoid pellet) - transfer to clean Eppendorf 1.5 ml tube
Add 400 µl of DNA/RNA lysis buffer (page 5, step 4) - close and invert gently to mix (end of page 5)
Yellow (DNA)
43m
transfer 700 µl of sample/lysis-buffer-mix to top of YELLOW DNA spin-away filter (100 µl will be left behind) (Page 7: (IV) DNA and RNA Purification)
Centrifuge YELLOW DNA column at 16,000x G for 30 seconds 16000 rpm, 00:00:30
30s
transfer flow through to a clean 1.5 ml Eppendorf tube (this contains RNA) and transfer YELLOW DNA column to a fresh collection tube (this contains DNA)
Add 700 ul of 100% ethanol to the flow-through in the eppendorf tube and invert to mix
Green (RNA)
add 700 µl of the ethanol-sample mixture to the top of the GREEN RNA column
Yellow (DNA)
43m
add 400 µl of DNA/RNA PREP buffer to the top of the YELLOW DNA column
Centrifuge all spin columns (green and yellow) and discard all flow through
Green (RNA)
add remaining 700 µl of ethanol-sample mixture (in eppendorf tube) to the top of GREEN RNA columns
Yellow (DNA)
43m
add 700 µl of DNA/RNA wash buffer to top of YELLOW DNA columns
centrifuge all columns (green and yellow) and discard all flow through 16000 rpm, 00:00:30
30s
BOTH
Add 400 µl of DNA/RNA wash buffer to both GREEN RNA columns & YELLOW DNA columns - place the YELLOWs and the GREENs into two separate centrifuge racks
Green (RNA)
43m
centrifuge the GREEN RNA columns for 30 seconds16000 x g, 00:00:30
30s
Yellow (DNA)
43m
centrifuge the YELLOW DNA columns for 2 minutes 16000 x g, 00:02:00
2m
Green (RNA)
Discard flow through from GREEN RNA columns.
Add 80 µl of DNase 1 reaction mix directly to each filter, but be careful not to touch or poke the filters.
Incubate 15 minutes 00:15:00
15m
Yellow (DNA)
6m
put YELLOW DNA column into a clean fresh collection tube and discard the old collection tube.
Add 50 µl ZymoBIOMICS DNase/RNase-Free water directly to the center of each YELLOW DNA column
(Again, be careful not to touch or poke the filters)
Incubate YELLOW DNA columns for 5 minutes
00:05:00
5m
Centrifuge YELLOW DNA columns 16000 x g, 00:00:30
30s
Transfer YELLOW DNA columns to clean eppendorf tubes (make sure caps are labeled)
Pipette flow-through (should be 50µl) back into the center of each respective column.
(again, be careful not to poke or touch the filter)
Centrifuge again (at lower speed to prevent breaking) 10000 x g, 00:00:30
30s
Replace any Eppendorf tubes that may have broken in the centrifuge.
Put DNA samples in the fridge (make sure labeled D for DNA along with the sample number)
Green (RNA)
7m 30s
After DNAse I 15-minute incubation is finished:
add 400 µl of DNA/RNA Prep buffer to top of GREEN RNA columns
Centrifuge (make sure to turn speed make up) 16000 x g, 00:00:30
30s
Discard Flow through
add 700 µl DNA/RNA wash buffer to top of GREEN RNA columns
Centrifuge 16000 x g, 00:00:30
Discard Flow through
add 400 µl DNA/RNA wash buffer to top of GREEN RNA columns
centrifuge for 2 minutes 16000 x g, 00:02:00
2m
Transfer columns to fresh collection tube and discard the old collection tube
Add 50 µl ZymoBIOMICS DNase/RNase-Free water directly to the center of each column
(Again, be careful not to touch or poke the filters)
Incubate for 5 minutes 00:05:00
5m
Centrifuge 16000 x g, 00:00:30
Transfer GREEN RNA columns to clean eppendorf tubes (make sure caps are labeled)
Pipette flow-through (should be 50µl) back into the center of each respective column.
(again, be careful not to poke or touch the filter)
Centrifuge again (at lower speed to prevent breaking) 10000 x g, 00:00:30
Replace any Eppendorf tubes that may have broken in the centrifuge.
Put RNA samples in the fridge (make sure they arelabeled R for RNA along with the sample number)
Qubit Quantify DNA
Collect 3 sets of strip tubes (Standard 1, Standard 2, and DNA)
Tear strip tubes if needed to have appropriate amount of samples (I.e. 6 samples = 6 tubes per set) and make note on the side of which row is which
Use DNA 1x BR working solution (in dark brown bottle) - calculate how much you need for all samples (200*19 = 3800 ul), transfer with pipette 3800 to plastic well
Pipette 190 µL of working solution to standard 1 and standard 2 with 8-channel, but 6 tips
Pipette 195 µL to DNA strip tubes using 8-Channel pipette, but 6 tips
Pipette 10 µL of Standard 1 into corresponding strip tubes using electronic pipette
Pipette 10 µL of Standard 2 into corresponding strip tubes using electronic pipette
Pipette 5 µL of each DNA into its corresponding strip tube using 2-20 µL pipette (red) set to 5
Vortex each strip for 3 seconds
Centrifuge each strip for 3 seconds
Incubate for 2 minutes
Select appropriate setting for DNA run (make sure it is DNA 1x BR (second page of menu)
Put in Standard 1 before selecting run (should appear in bottom left corner)
Put in Standard 2 before selecting run (Should appear in top right corner)
Put in DNA strip before selecting run
Record data
Qubit Quantify RNA