Mar 02, 2020
Version 2
Sterivex DNA extraction V.2
- Ariel Rabines1,
- Rob Lampe1,
- Andrew E Allen1
- 1Scripps Institution of Oceanography
- A.E. Allen Lab
Protocol Citation: Ariel Rabines, Rob Lampe, Andrew E Allen 2020. Sterivex DNA extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.bc2hiyb6
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: Working
We use this protocol and it's working
Created: February 26, 2020
Last Modified: March 02, 2020
Protocol Integer ID: 33577
Keywords: sterivex, DNA, epMotion, automated, extraction, environmental, seawater, marine, ocean,
Abstract
A mostly automated protocol for extraction of genomic DNA from seawater filtered onto a Sterivex filter (Cat. No. SVGP0150). Reagents come from the Macherey-Nagel NucleoMag Plant Kit for DNA purfication (Cat. No. 744400). Automated liquid handling is performed on an eppendorf EpMotion 5075t with multi-channel pipettes. This protocol assumes that the Sterivex is sealed with tube sealant on the male end and a male-luer lock plug on the female end. Using this protocol, we routinely extract ~80 samples at a time.
The protocol has been modified to increase to an 800 µL starting volume. This normally results in less Lysis Buffer MC1 and Binding Buffer MC2 than is needed. Extra binding buffer should be purchased separately.
The kit manual can be found here: https://www.mn-net.com/media/pdf/09/46/a0/Instruction-NucleoMag-Plant.pdf
Additional notes from the manufacturer on using the kit with the epMotion can be found here: https://www.mn-net.com/media/pdf/95/2c/ef/AN-NucleoMag-Plant-epMotion5075.pdf . Note that we have modifications to their protocol.
The epMotion program is attached here.
Attachments
Guidelines
A mostly automated protocol for extraction of genomic DNA from seawater filtered onto a Sterivex filter (Cat. No. SVGP0150). Reagents come from the Macherey-Nagel NucleoMag Plant Kit for DNA purfication (Cat. No. 744400). Automated liquid handling is performed on an eppendorf EpMotion 5075t with multi-channel pipettes. Importantly, the epMotion must be equipped with a gripper, 1000 uL multichannel, 300 uL multichannel, an integrated themomixer, an a thermomodule. This protocol assumes that the Sterivex is sealed with tube sealant on the male end and a male-luer lock plug on the female end. Using this protocol, we routinely extract ~80 samples at a time.
The protocol has been modified to increase to an 800 µL starting volume. This normally results in less Lysis Buffer MC1 and Binding Buffer MC2 than is needed. MC2 is increased for the bead/MC2 mixture while the beads volume is kept the same. Extra binding buffer is sold in 1L quantities and should be purchased separately.
Materials
MATERIALS
80% Ethanol
MAGNUM EX Universal Magnet PlateAlpaquaCatalog #A000380
Masterblock 96 Deep Well Plategreiner bio-oneCatalog #780286
Flexible Tube CutterCatalog #97642
Vortex Adapter for 5mL tubesMobioCatalog #13000-V1-5
twin.tec® PCR plate 96 LoBind skirted 150 µL PCR cleanEppendorfCatalog # Catalog No.
Before start
- Prepare extraction sheet listing sample name, extraction number (starting from #1), and plate position (e.g. A1). We normally randomly add a few blank samples (no liquid in starting deep well plate) scattered throughout the positions.
- Clean all surfaces and tools with 70% EtOh.
- Set shaker to 56 °C
Lysis
Lysis
Prepare DNA lysis buffer (N = number of samples plus extra for pipetting)
In sterile container, add 800 µL x N of Lysis Buffer MC1
Add 10 µL x N RNase A
(optional) Add 1 µL x N of each internal standard
Mix well.
Keep sterivex on dry ice. In each, remove luer plug and pipette the per sample volume prepared in step 1 into each. For example, if 800 µL MC1 + 10 µL RNase A + 2 µL internal standards were used, add 812 µL . Dispense quickly as the lysis buffer will freeze upon contact. Replug with luer lock. Place luer-side down into 1.5 mL tube rack.
Incubate at 56 °C and shake gently for 00:30:00 .
Vortex for 00:05:00 using adapter for 5 mL tubes.
Transfer lysate to sterile 1.5 mL tubes labeled with extraction numbers.
Label each sterviex with its extraction number.
Pop the lid with the male end off the end of the sterivex using the tube cutters. The tube cutters will fit in the lip between the lid and the rest of the unit. Apply pressure at an upward angle and it should release. Continue to cut around the top if needed.
Rub the filter on the inside of the sterviex to release as much of the lysis buffer and material as possible. Then pipette as much liquid as possible from inside the sterviex into the corresponding 1.5 mL tube.
Clear lysate by centrifugaion at 5600 x g, 00:20:00 .
Note
This is a good time to prepare your buffers. Start the program on the epMotion which will calculate your buffer volumes depending on the number of samples. Add a few mL more than is requested and enter that as the volume supplied. We premix the beads and Binding Buffer MC2 at a ratio of 30 µL beads:770 µL MC2. Note that this keeps the bead amount the same as specified in the manual while increasing the amount of MC2.
Transfer to another set of numbered 1.5 mL tubes. Remove as much supernatent as possible without disturbing the pellet.
Align 1.5 mL tubes in rack in plate position. Set adjustable spacer multichannel pipette to 800 µL and transfer no more than 800 µL into the deep well plate.
Transfer deep well plate to the epMotion and run program. The final elution is always into a twin.tec LoBind PCR plate (see materials).
Note
Reservoir rack layout (left to right):
- MC2 with beads
- MC3
- MC4
- 80% EtOH
- MC5
- MC6
Note
Step 2 in the program stops it. At this point with a P1000 multichannel pipette, throughly pipette up and down to mix the bead solution then continue the run.
Once program is complete, run 2.5 µL of the extracted DNA on a gel. Good samples will show a bright band of high molecular weight DNA. Blank samples should show nothing. Degraded samples will show a smear.
Note
We normally use of a 1:10 dilution of this extracted DNA for our template in PCRs for amplicon library preperation. This is a good time to also make the 1:10 dilution plate (20 µL total volume) using the elution buffer MC6 (5 mM Tris/HCl, pH 8.5).