Methods – DNA Extraction (if required)
DNA extraction protocols have been optimised for each taxon group processed at the MBA to ensure effective and efficient tissue lysis and homogenisation, and to generate high-quality,
high-yield template DNA for PCR amplification of DNA barcoding loci.
Samples are typically processed in batches of 8-12, organised according to extraction needs. For example, tough brown macroalgae (phaeophytes) that are expected to require a lot of
troubleshooting are processed in smaller batches.
During DNA extraction and barcoding processes, a simple numbering scheme is used to label tubes, rather than using the full DToL sample numbers. This is tracked and recorded in the
lab book. Resultant DNA extracts should be labelled with the full DToL sample
numbers.
DNA extraction of macroalgae (if required)
This is a modified version of the Nucleospin Plant II kit (Macherey Nagel) Protocol 5.1 Genomic DNA from plants. This should also be followed for lichens. Read manufacturer’s instructions alongside
these modifications thoroughly before commencing work. Refer to manufacturer’s
protocol Section 2.5 for choosing the optimal lysis buffer for your sample.
1. Transfer tissue from FluidX tube to a bead-beating tube, pre-filled with approximately
half a PCR tube (0.2ml) of 2mm Zirconium beads, using sterilised needle or
pipette tip, if not done so already. Retain samples on dry ice to avoid
thawing.
2. Grind in Bead Beater for 40s x 6 ms, and return to dry ice for 5 mins.
Inspect samples. Add 2 x 3mm Tungsten carbide beads to samples that have not degraded at all.
Grind in Bead Beater for 40s x 8 ms, and return to dry ice for 5 mins.
Inspect samples. For those with no visible degradation, complete freeze-thaw cycles of freezing in liquid nitrogen for 30 s and thawing at 65°C.
Final cycle with 400 ml Buffer PL1 (step 2a) but not RNase A
If samples are not homogenised, repeat cycle 40s x 6 ms or if there is no
visible degradation, follow ‘Trouble-shooting cycles’ as necessary.
3. Proceed with cell lysis using Buffer PL1 (step 4a) or alternatively Buffer PL2 (step 4b).
4a. Add 400µl Buffer PL1. Vortex the mixture thoroughly. Add 10µl RNase A solution and invert. Incubate the suspension for 1-2 hours at room temperature.
• Trouble-shooting: inspect samples, if they are particularly viscous, add another 400µl PL1 and 4µl RNase A. Vortex to mix.
4b. Transfer the resulting powder to a new tube and add 300µl Buffer PL2. Vortex the mixture thoroughly. Add 10µl RNase A solution and invert. Incubate the suspension for 1-2 hours at room temperature.
• Trouble-shooting: inspect samples, if they are particularly viscous, add another 300µl PL2 and 4µl RNase A. Vortex to mix.
Add 75µl Buffer PL3, mix thoroughly and incubate for 5 mins on ice to precipitate SDS completely. If additional PL2 has been added, add a further 75µl Buffer PL3.
5. Centrifuge samples for 2 mins at 13,000 x g.
6. Place a NucleoSpin Filter (violet ring) into a new collection tube (2 ml) and load the lysate onto the column, taking care to avoid the pellet.
7. Centrifuge for 2 mins at 11,000 x g, collect the clear flow-through and discard the NucleoSpin Filter. If not all the liquid had passed the filter, repeat the centrifugation step.
8. Taking care to avoid any pellet, transfer the clear supernatant to a new 1.5ml microcentrifuge tube (not provided).
9. Add 450µl Buffer PC and mix thoroughly by pipetting up and down (5 times) or by vortexing
a. For samples that have additional lysis Buffer PL1 or PL2/PL3, add the same proportion of Buffer PC.
10. Place a NucleoSpin Plant II Column (green ring) into a new Collection Tube (2ml) and load a maximum of 650µl of the sample.
a. According to manufacturer’s instructions, the maximum loading capacity of the NucleoSpin Plant II Column is 700µl but it is not possible to then close the lid. Loading between 600-650µl is recommended. For higher sample volumes, repeat the loading step, by loading lysate and centrifuge for 1 min at 11,000 x g.
11. Centrifuge for 1 min at 11,000 x g and discard the flow-through.
12. Add 400µl Buffer PW1 to the NucleoSpin Plant II Column. Centrifuge for 1 min at 11,000 x g and discard flow-through.
13. Add 700µl Buffer PW2 to the NucleoSpin Plant II Column. Centrifuge for 1 min at 11,000 x g and discard flow-through.
14. Add another 200µl Buffer PW2 to the NucleoSpin Plant II Column. Centrifuge for 1 min at 11,000 x g. Discard flow-through and centrifuge for 2 mins at 11,000 x g in order to removed wash buffer and dry the silica membrane completely.
15. Place the NucleoSpin Plant II Column into a new 1.5ml microcentrifuge tube (not provided). Pipette 50 l DNase-free water (65C) onto the membrane. Incubate the NucleoSpin Plant II Column for 5 minutes at 65C. Centrifuge for 1 min at 11,000 x g to elute the DNA.
a. If a more dilute sample is desired, repeat this step with another 50µl DNase-free water (65C) and elute into the same tube.
16. Using the Nanodrop, record DNA quantity and quality (noting ng/µl, 260/230 and 260/280 values), in Macroalgae Progress log in the Barcoding folder on the DToL shared drive.
a. If the DNA quality is poor and the sample is < 10 ng/µl or > 50 ng/µl, additionally assess quality using the Qubit HS dsDNA kit.
17. Store DNA in 1.5ml microcentrifuge elution tube at -20C for short term storage whilst confirming identity using barcoding. Once sent for genome sequencing, transfer to storage at -80C.
DNA extraction of protists (microalgae) (if required)
Follow recommendations outlined in the DToL for Protist Sample Preparation SOP, using a DNA extraction kit e.g. DNeasy Plant kit (Qiagen) and following the manufacturer’s instructions. Record DNA yield and quality using Nanodrop and/or Qubit HS dsDNA kit in Protist/Microalgae Progress log in the Barcoding folder on the DToL shared drive. Store DNA in 1.5ml microcentrifuge elution tube at -20 C for short term storage whilst confirming identity using barcoding. Once sent for genome sequencing, transfer to storage at -80 C.
DNA extractions of fungi and lichens (if required)
For fungi, this follows the MBA Fungal Culture Collection DNA extraction protocol (dated 24.06.2020) using DNeasy Plant kit (Qiagen).
If sample is very small, the DNA extraction protocol for macroalgae described earlier should be followed. Otherwise continue with liquid nitrogen grinding, as described below.
1. Pre-cool a pestle and mortar by pouring liquid nitrogen into the mortar. Pre-cool a spatula and an Eppendorf tube for the ground sample in liquid nitrogen.
2. Place the frozen sample in the dry ice – don’t let it defrost. Pour on some liquid nitrogen and grind the sample in the liquid.
3. Continue grinding until the sample is a fine powder. Add more liquid nitrogen between grinding if needed.
4. Using the pre-cooled spatula, scrape the powder off the pestle and mortar and place it in an Eppendorf tube.
5. Add 400µl of PL1 buffer from the NucleoSpin Plant II DNA extraction kit (Macherey Nagel) and vortex sample. Continue DNA extraction by following steps 4-17 in macroalgae DNA extraction.
1. If not already prepared, resuspend pellet or scraped biomass in FluidX tube with molecular-grade water and transfer to bead-beating tube, prefilled with a mixture of glass and zirconium beads (100 µm – 2 mm). Centrifuge at 4000 rpm for 5 minutes to pellet, remove supernatant.
2. Bead beat for 1 min at 10ms with 5 mins rest on ice, repeat three times.
3. Transfer lysate (liquid) into new sterile 1.5ml tube.
4. Centrifuge at max speed (13,000 rpm) for 5 mins, discard supernatant (liquid).
5. Add 400µl Buffer AP1 and 4µl RNase A. Vortex and incubate for 10 mins at 65C. Invert 3 times during incubation.
6. Add 130µl Buffer P3. Mix and incubate for 5 mins on ice.
7. Centrifuge lysate for 5 mins at maximum speed (13,000 rpm).
8. Pipette lysate into QIAshredder spin column placed in a 2 ml collection tube. Centrifuge for 2 mins at maximum speed (13,000 rpm).
9. Transfer the flow-through into a new tube without disturbing the pellet (if present). Add 1.5 volumes of Buffer AW1 and mix by pipetting.
10. Transfer 650µl of the mixture into a DNeasy Mini spin column placed in a 2 ml collection tube. Centrifuge for 1 min at 8,000 rpm. Discard flow-through. Repeat this step with the remaining sample.
11. Place the spin column into a new 2 ml collection tube. Add 500µl Buffer AW2 and centrifuge for 1 min at 8,000 rpm. Discard flow-through.
12. Add another 500µl Buffer AW2. Centrifuge for 2 mins at maximum speed (13,000 rpm)
a. Remove the spin column from the collection tube carefully so that the column does not come into contact with flow-through.
13. Discard the flow-through and collection tube. Place spin column into a new 1.5 ml microcentrifuge tube.
14. Add 100µl Buffer AE (pipette directly onto DNeasy membrane) and incubate at room temperature for 5 mins.
a. Elution volume can be reduced (e.g. to 50µl) to increase yielded DNA concentration. This will reduce overall yield.
15. Centrifuge for 1 min at 8,000 rpm.
16. Using Nanodrop, record DNA quantity and quality (noting ng/µl, 260/230 and 260/280 values), in Fungi Progress log in Barcoding folder on DToL shared drive.
17. Store DNA in 1.5ml microcentrifuge elution tube at -20 C for short term storage whilst confirming identity using barcoding. Once sent for genome sequencing, transfer to storage at -80C.