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

[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 and lichen 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 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: Lichens

Including: 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.  

Lichen barcoding for the Darwin Tree of Life (DToL) project involves extraction of DNA from lichen material that has been flash-frozen or dried and then frozen, 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. Field collection and sampling of lichens, both in the field and in the lab, follows the Collections Standard Operating Protocol: Lichens.  Different extraction protocols are used depending on lichen morphology and specimen size. 


 

SECTION: DNA extraction (Qiagen DNeasy plant mini kits)
2. DNA extraction using acetone rinse with REDExtract-N-AmpTM

 

Acetone rinse (to reduce lichen secondary compounds)
1. Place the lichen fragment(s) in a 1.7 ml tube.
2. Soak the sample in c. 50 μl acetone for at least 30 min, ensuring that the tissue is completely covered by the acetone. 
3. Pipette off the acetone into a labelled 500 μl tube. 
4. Leave the lichen tubes and the acetone tubes open in a fume hood to air dry (c. 10 min for lichen tubes, and 30 min-1 hour for acetone tubes). 
5. Once dry, either pass the acetone sample tube on for thin layer chromatography (TLC), or store it with the lichen herbarium specimen in case TLC will be carried out in the future. Carry on with DNA extraction of the lichen sample tubes.

DNA extraction
1. Aliquot REDExtract-N-AmpTM reagents into 1.5 ml aliquots and store at -20°C until needed.
2. Preheat a heat block to 95°C (c. 20 min) and defrost aliquots of REDExtract-N-AmpTM extraction and dilution solutions.
3. Add c. 30 μl extraction solution to each lichen sample to cover.
4. Vortex, spin down and ensure lichen tissue is well submerged. 
5. Incubate for 10 min at 95°C (keeping an eye on the lids, as they may pop open).
6. Add 30 μl dilution solution to neutralize inhibitors (the same volume as extraction buffer).
7. Store at 4°C until needed.    

 

Dilution of DNA stock for PCR and sequencing
In PCR strips, dilute DNA stocks with molecular grade water to a 1:10 dilution (e.g. 3 μl DNA + 27 μl water) for standard samples. Smaller (just visible/scant samples) can be diluted 1:5 (3 μl DNA + 12 μl water).

SECTION: DNA extraction (Qiagen DNeasy plant mini kits)
3. DNA extraction using Bento Dipstick DNA Extraction Kit

 

1. Add a pinch of sterilised sand and a small amount of extraction buffer (c. 20-40 μl) to lichen sample tubes; homogenize well with a micro pestle.
2. Add additional buffer incrementally (for a total of c. 300 μl for very small samples—recommended adding up to 500 μl, depending on the amount of material); homogenize further as needed.
3. Prepare a wash buffer tube for each lichen sample. Recommended volume is 750ul in a 1.7 ml tube (500 μl for small samples).
4. Prepare 25 μl TE buffer (Tris 10 mM EDTA 1 mM) in a labelled 0.5 ml tube for each sample (to make a DNA stock).
5. On a clean piece of paper or foil, organize 2 dipsticks for each sample.
6. With the 3 prepared tubes for each sample lined up (DNA extract, wash buffer, TE buffer):
     a. Dip the cellulose end of a dipstick into the DNA/extraction buffer 3 times to capture DNA.
     b. Dip the dipstick into the wash buffer 3 times (pressing the cellulose end into the side of the tube to 
     squeeze off excess wash buffer).
     c. Dip the dipstick into the TE buffer for c. 10 seconds, agitating gently, to release the DNA and make a                    
     stock. 
     d. Repeat the process for the second dipstick.
7. Spin down eluted DNA and store at -20°C long term, or 4°C for immediate use.
  

 

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

Frozen lichen sample material -This should be kept frozen in labeled batches, with specimens only left at room temperature long enough to sample for DNA extraction.
 
TE-preserved lichen material - Occasionally, fresh lichens are preserved in TE buffer and frozen for future DNA extraction. If any lichen thallus is left after the extraction process, return the tubes to the freezer drawer, in case extractions have to be repeated. 

DNA - Long term banking into a freezer is only necessary after all PCR from that extraction set is complete, including rePCRs of problematic material. 
 

SECTION: DNA extraction (Qiagen DNeasy plant mini kits)
4. DNA extraction with direct PCR

 

1. Prepare a 0.2 ml tube with a 20 μl PCR mix for each specimen (ideally 2 tubes per specimen).  
2. Working under a stereo microscope and with sterilised blades/needles, make sections of target lichen  tissue areas (e.g. slices of apothecia/perithecia).  
 3. Working on a slide (and under higher magnification), make a dissection of the section to isolate target tissue for direct PCR (e.g. hymenium only).  
 4. Using a sterile pipette tip or needle (moistened in sterile water or PCR mix), pick up a minute amount of target material. Add the piece of lichen to the PCR tube, ensuring the material arrives into the tube (this may require examination of the tube under the microscope).  
5. Carry out PCR.