TITLE: DNA Barcoding Standard Operating Protocol, Plants and Lichens at RBGE, Lab methods: DNA extraction
AUTHORS: Laura L Forrest, Rebecca Yahr, Michelle Hart

[DESCRIPTION]
This is part of the collection DToL Taxon-specific Standard Operating Procedures for the Plant Working Group (protocols.io). The SOP collection contains guidance on how to process the various land plant taxa within the scope of the Darwin Tree of Life project. The guidance specifically refers to the tissue samples needed for DNA barcoding (which takes place at the Royal Botanic Garden (RBGE)) . Every specimen is submitted for DNA barcoding first before potentially being sent to the Wellcome Sanger institute.

This DNA barcoding SOP outlines DNA extractions from plant and lichen samples for the Darwin Tree of Life project (DToL) at the Royal Botanic Garden Edinburgh (RBGE). 

DNA barcoding is used as part of the species identification process AND sample tracking (to check that the genome sequence corresponds to the material that was sent and that there have been no sample mix-ups).

Definition: Land plants (Embryophyta) and lichens
Including: Bryophyta, Marchantiophyta, Anthocerotophyta, Lycopodiophyta, Polypodiophyta, Pinophyta, Cycadophyta, Ginkgophyta, Gnetophyta, Magnoliophyta, lichenized fungi

Excluding: all non-lichenized fungi



[GUIDELINES]

Including: Marchantiophyta (liverworts), Bryophyta (mosses), Anthocerotophyta (hornworts), vascular plants, lichens


 






 
     Version      Date      Major changes      Contributors        1.0      2020-04-07      Draft   version      Lyndall   Pereira (NHM)        2.0      2020-04-29      DNA   barcoding for reference library at NHM edited      NHM: Olga   Sivell, Laura Sivess, Gavin Broad, Ben Price, Lyndall Pereira        3.0      2020-05-13      Addition of   pictorial dissection for each taxon group      Liam   Crowley (Oxford WW) and Lyndall Pereira (NHM)        3.1      2020-07-17       SOP text   split into Order-specific SOPs. Uniform style, publication quality images   with colour blind friendly dissection lines      Olga   Sivell, Laura Sivess        4.0      2022-01-04      Overall   formatting further improved (including spelling/ punctuation changes, table   of contents links), photo guides made more uniform, edits in protocol made to   reflect any adaptations in the sampling process since July 2020, additional   taxon protocols included, unrequired taxon guides removed. First submission   to protocols.io.      Inez   Januszczak (NHM), Liam Crowley (Oxford WW), Olga Sivell (NHM), Laura Sivess   (NHM), Chris Fletcher (NHM)   



 Working SOP, checked by experts
 

[STEPS]
SECTION: DNA extraction (Qiagen DNeasy plant mini kits)
1. Sampling.  

Plant DNA barcoding for the Darwin Tree of Life (DToL) project involves extraction of DNA from silica gel dried tissue that has been provided, along with the required metadata, following the collecting and submission SOPS available in the collection DToL Taxon-specific Standard Operating Procedures for the Plant Working Group (protocols.io) 
 



SECTION: DNA extraction (Qiagen DNeasy plant mini kits)
2. Homogenization.

Plants. 
On a vented bench, using clean forceps, put around 1-2 cm2 silica-gel dried (or herbarium) plant material into a labelled open-based Axygen 2 mL microcentrifuge tube, along with a flattened tungsten bead. 
Distribute the tubes or plates between two balanced adaptors. 
Homogenize for 2 mins at 20 Hz, rotate the adaptor 180°, then homogenize for a further 2 mins at 20 Hz. 
If the sample has ground poorly, add a second flattened bead and repeat.
If problems persist, transfer the sample or a new bit of plant tissue into a 1.7 ml eppendorf tube, add a pinch of acid-washed sand, and manually grind using a disposable plastic mini-pestle.
 
Lichens. 
Place a small fragment of lichen into the bottom of a 1.7 ml eppendorf tube (if grinding with a mini-pestle) or a 2 ml eppendorf tube (if grinding with a mixer mill).
Soak the lichen tissue in c. 50 μl acetone for at least half an hour, ensuring that the tissue is completely covered by the acetone. 
Pipette off the acetone into a labelled 500 μl tube. 
Leave the lichen tubes and the acetone tubes open  in a fume hood to air dry. 
Once dry, grind the sample using either a disposable plastic mini-pestle and a little acid-washed sand, or a mixer mill and 1-2 flattened tungsten beads (following the plant protocol above).
Either pass the air-dried acetone sample on for thin layer chromatography (TLC), or store it with the lichen herbarium specimen in case TLC will be carried out in the future.
 

SECTION: DNA extraction (Qiagen DNeasy plant mini kits)
3. DNA extraction using DNeasy® Plant mini kits (QIAGEN): 

Follow manufacturer’s protocols, but: 
Extend the incubation period in lysis buffer to an hour at 65°C in a thermomixer block set to 800 RPM. 
Optionally, extend the on-ice incubation (this is a good point for a lunch break) - a longer incubation can help with polysaccharide-rich tissues. 
Avoid using too much starting material, as this can block the DNeasy column membrane. It is particularly important not to overdo starting material from mucous-rich plants. 
Always centrifuge for 5 minutes at full speed after the first incubation (this is an optional step in the DNeasy manufacturer’s protocol) to pellet the homogenized material. 
Use filter tips whenever transferring or mixing liquids with DNA in them.
 
A double elution, with 75 μL pre-heated 65°C QIAGEN elution buffer at the first step and 50 μL pre-heated 65°C QIAGEN elution buffer at the second step, is recommended, giving a final extraction volume of c. 100 μL.
 

SECTION: DNA extraction (Qiagen DNeasy plant mini kits)
4. Storage 

Silica gel dried plant tissue – this should be stored out of direct light, in a temperature and humidity controlled environment.
 
TE-preserved lichen material – if any lichen thallus is left, return the tubes to the freezer drawer, in case extractions have to be repeated. 

DNA -  as we elute in a buffer that contains EDTA, the DNA can be stored at 4°C for easy access and to avoid freeze-thaw cycles. Long term banking into a freezer is only necessary after all PCR from that extraction set is complete, including rePCRs of problem material.