Feb 27, 2020
Plant and fungal DNA extraction using a QIAGEN QIAxtractor
- 1Royal Botanic Garden Edinburgh
Protocol Citation: Laura L Forrest, Michelle Hart, Alexandra Clark 2020. Plant and fungal DNA extraction using a QIAGEN QIAxtractor. protocols.io https://dx.doi.org/10.17504/protocols.io.bc2fiybn
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 27, 2020
Last Modified: February 27, 2020
Protocol Integer ID: 33575
Keywords: Bryophyte, DNA extraction, DNA barcoding, RBGE, QIAcube, QIAxtractor, angiosperm, fungi
Abstract
Here we describe a semi-robotic plate DNA extraction method that we have used at the Royal Botanic Garden Edinburgh (RBGE) to process samples for PCR and Sanger sequencing. This protocol was developed at RBGE in conjunction with QIAGEN for DNA extraction from a broad range of plant and fungal tissue.
The RBGE QIAxtractor has been updated to a QIAcube; this protocol remains the same.
Guidelines
Ensure sampling is compliant with the Nagoya Protocol (Buck & Hamilton, 2011), which came into force on the 12th October 2014
Materials
| Corbett Cat | QIAGEN Cat | QIAGEN Name | ||
| Consumable Pack, QIAxtractor | 2270 | 950007 | Consumable Pack, QIAxtractor | |
| Plastic ware Pack, DX, QIAxtractor | 2260 | 950017 | Plastic ware Pack, DX, QIAxtractor | |
| Lysis Plate, 96-well (5) | 2271 | 990600 | Lysis Plate, 96-well (5) | |
| Capture Plate, standard yield (5) | 913 | 950901 | Capture Plate, standard yield (5) | |
| Elution Plate (10) | 2147 | 990602 | Elution Plate (10) | |
| Elution Plate 8-Strip Caps (120) | 1636 | 990604 | Elution Plate 8-Strip Caps (120) | |
| Capture Plate, DX (5) | 2273 | 950911 | Capture Plate, DX (5) | |
| Reagent Pack, DX | 2220 | 950107 | Reagent Pack, DX | |
| Tissue Digest Reagent (DXT) (250 ml) | 2228 | 950183 | Tissue Digest Reagent (DXT) (250 ml) | |
Spin the homogenized tissue samples, in plates of 1.2 ml QIAGEN collection microtubes, for c. 10 mins at full speed in order to remove as much of the loose tissue powder as possible from near the tube lids. If only extracting one plate of samples instead of two, the centrifuge can be balanced by shifting half the strips of microtubes into a new rack (alternate strips and empty rows across the racks).
Lysis pre-treatment: in a trough mix 4 μl DX enzyme (kept at 4° C in the fridge) and 416 μl Tissue Digest (DXT) per sample (master mix 40.4 ml DXT and 392 μl DX enzyme per 96-sample plate); if extracting from fresh tissue rather than silica-dried or herbarium samples, substitute 4 μl DXT with 4 μl RNase A.
Separate each row of strips by at least one empty row before removing lids to mimimize the possibility of contamination. Carefully remove the lids from the strips, avoiding transferring any loose powder between tubes or contaminating gloves. Using a multichannel pipette, add 400 μl of the lysis mix to the centrifuged homogenized tissue samples in the 1.2 ml QIAGEN collection microtubes, shake the tubes to collect any powder from the lids and sides, then incubate for 1 hr to 1 ½ hrs in an Eppendorf ThermoMixer, increasing from room temperature to 70° C, with 700-800 rpm shaking.
Centrifuge plate of 1.2 ml QIAGEN collection microtubes at maximum speed for 10 min to pellet the sample debris. With a single plate, ensure centrifuge is fully balanced by splitting the strips of microtubes across two racks.
Using a multichannel pipette, transfer 220 μl of each sample from the plate of 1.2 ml QIAGEN collection microtubes into a 96-well Lysis Plate, avoiding disturbing the pellets.
Remove the tungsten beads that were used to homogenize the samples from the collection microtubes, and clean them for reuse: Flush with running water, sonicate in Ultraware ultrasonic bath for c. 2 mins to remove any stray sample fragments from the beads, then incubate in 0.4 M Hydrochloric acid for 1 min. Flush again with running water to remove the acid, and air dry.
Put the Lysis plate into the QIAxtractor.
In the machine, per plate of 96 samples, also add 66 ml DX Binding (DXB) (kept in the fridge at 4° C), 122 ml DX Wash (DXW), 62 ml DX Final Wash (DXF) and 12.4 ml Elution buffer (E).
The machine has been programmed to robotically add 440 μl DXB to each 220 μl sample in the Lysis Plate and incubate for 5 min. It then transfers 600 μl of this mixture into the vacuum plate, with a 5 min vacuum to pull the liquid through and dry the membrane. The 4 wash steps involve: 200 μl DXB followed by a 5 min vacuum, 600 μl DXW followed by a 1 min vacuum, another 600 μl DXW followed by a 1 min vacuum, then 600 μl DXF followed by a 1 min vacuum, followed by a 5 min vacuum. The vacuum plate is transferred to above an elution plate, and 100 μl DXE is added and incubated for 5 min, followed by a 1 min vacuum to elute the DNA from the membranes.
Transfer the extracted DNA from the elution plate into new labelled plates or tubes, and store at -20° C or 4° C.