A cost-effective way of extracting high molecular weight (HMW) DNA from low amounts of fresh or herbarium lichen material

If necessary clean lichen material using nuclease-free water and dry the sample* Cut lichen material into small pieces (µm) using a sterile razor blade or scalpel

• Transfer samples into 2.0 ml Eppendorf Protein LoBind tubes and store them at -80°C
• Add 400 µL cold acetone (stored at -20°C), shake gently and remove the supernatant. Supernatant can be stored for use in thin-layer chromatography (TLC)
• Repeat until supernatant is clear
• Put the tubes in a heating block set at 30°C under the fume hood until the samples are dry

Buffer 1 : prepare 1 mL/sample (plus overhang) using liquid stocks

• 100 mM Tris-HCl
• Add 10 mM DTT solution just before use

Buffer 2 : prepare 500 µL/sample (plus overhang) under the fume hood using liquid stocks

• 1 M Sorbitol
• 25 mM EDTA
• 5.9 mM Citric Acid
• 4.1 mM Sodium Citrate
• 14 mM ß-Mercaptoethanol

Add 1 mL Buffer 1 to each sample * Mix thoroughly by flipping the tube

• Incubate for 15 minutes at RT
• Centrifuge at 3000x g,0h 0m 0s for 2 minutes. If necessary repeat at a higher speed (e.g. for crust lichens) until a precipitate forms
• Discard supernatant
• Add 500 µL Buffer 2 to each sample
• Mix enzymes well before taking an aliquot. Then add 5 µL each of Glucuronidase , Cellulase , Protoplast F , and Lysozyme to each sample
• Add 90 µL Chitinase to each sample
• Place the tubes in a thermomixer overnight at 35°C and rotate at 750rpm for 9 seconds every 5 minutes

Set thermomixer to 56°C* Centrifuge samples with 3000x g,0h 0m 0s for 2 minutes

• Discard supernatant while working under the fume hood
• Add 600 µL Buffer ATL and mix slowly and carefully
• Add 20 µL Proteinase K and mix slowly and carefully
• Incubate in a thermomixer at 56°C with shaking at 600rpm for at least 1 hour
• Remove samples and set the thermomixer to 70°C
• Briefly centrifuge tubes to remove drops from inside the lid
• Add 600 µL Buffer AL , close the lid, and mix gently using the lab rocker at 5rpm, or by light shaking. Sample and Buffer AL have to be thoroughly mixed to yield a homogeneous solution
• For low-input samples, an optional step is to dissolve 1 µg of carrier RNA in 1 µL of buffer ATL . Then, add 1 µL of the dissolved carrier RNA to each sample
• Incubate tubes at 70°C in a thermomixer with shaking at 600rpm,0h 0m 0s for 10 minutes
• Briefly centrifuge tubes to remove drops from inside the lid
• Add 300 µL ethanol (96-100%), close the lid and mix by light shaking or on the lab rocker at 5rpm,0h 0m 0s for at least 10 seconds. Mix samples and ethanol thoroughly to ensure efficient binding
• Briefly centrifuge tubes to remove drops from inside the lid
• Transfer 700 µL of the supernatant into a labelled 1.5 mL DNA LowBind tube. Transfer the remaining ~800 µL into a second labelled tube, resulting in two storage vessels per sample

Thoroughly shake the SPRIselect bottle to resuspend the SPRI beads* Add *1.2 SPRIselect to the sample (sample volume [µL] * 1.2 = SPRIselect [µL])

• For long-read sequencing, an optional step is to add * 0.65 of PEG-NaCl-Buffer (sample volume [µL] * 0.65 = PEG-NaCl-Buffer [µL])
• Mix samples gently using the lab rocker, or rock the storage vessels from side to side and incubate at RT for 30 seconds. Mix well, as insufficient mixing will lead to inconsistent results.
• Repeat the mixing and incubating step
• Place tubes into the magnetic stand and allow the SPRI beads to settle to the magnets (settle times will vary)
• Remove the clear supernatant. Be aware that significant bead loss will result in reduced yield
• Add 700 µL 5 M NaCl without removing the tubes from the magnetic stand, incubate for 1 minute, and discard the supernatant
• Repeat the previous washing step
• Remove the tubes from the magnetic stand and add ≥ 20-70 µL of nuclease-free water (or standard buffer like Tris or TE)
• Carefully resuspend the beads by slowly mixing them on the lab rocker or by pipetting ten times using wide-pore pipettes until homogeneous
• Incubate at RT for 1 minute
• Place the tubes on a magnetic stand, allowing the SPRI beads to settle to the magnets. If the elution volume is too low so that beads cannot settle to the magnet, flip the magnetic stand while holding the reaction vessels in place
• Transfer the eluate (HMW DNA) into a labelled 1.5 ml DNA LowBind storage vessel, and combine divided samples
• Check DNA quantity and fragment size. If DNA fragments are too short, repeat the size selection part using *0.8 SPRIselect and * 1.0 PEG-NaCl-Buffer

Prepare 20%-PEG-0.75-M-NaCl -Buffer:

NaCl 58,44 g/mol: m = M * c * V

PEG 20% : V% = (v/v)% = volume of solute/volume of solution * 100

20 % = 20 mL/100 mL = x mL / 200 mL * 100

x = 20 mL * 200 mL / 100 mL = 40 mL PEG in 200 mL

Shake vigorously, vortex and shake again.