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Science Education

Formerly known as European Learning Laboratory for the Life Sciences

Our inspiring educational experiences share the scientific discoveries of EMBL with young learners aged 10-19 years and teachers in Europe and beyond. We belong to EMBL’s Science Education and Public Engagement office.

Sample collection and wet-lab protocols

Required materials

The following protocols give instructions on collecting plant samples and detail two alternative ways of extracting, amplifying and visualising DNA barcoding marker genes. It also provides advice on Sanger sequencing.

Materials required for wet-lab activity

The following section contains information on the equipment and consumables required for running the sample collection and wet-lab part of plant DNA barcoding.

We describe two alternative protocols for DNA extraction, amplification and visualisation, a “direct PCR” protocol and version which uses spin-columns. Alternative methods not described here might also be suitable.

Direct PCR protocol

General equipment

  • Micropipettes (for a range of 1 to 200 μl; ideally P2, P20, P200)
  • Micropipette tips, sterile
  • 1.5 ml Eppendorf tubes, sterile
  • Optional: Vortex mixer
  • Table-top centrifuge (speed 20,000 x g)
  • Eppendorf racks
  • Bench waste containers for small waste such as tips
  • Ice buckets and ice to keep samples cold
  • Scissors
  • Permanent marker pens to label tubes

Plant sample collection

  • Collection containers (e.g. 15 ml Falcon tubes), one per sample
  • Sample collection record sheets (download), one per two samples
  • Optional: smartphone to take pictures of samples and record their GPS locations

DNA extraction and PCR

This describes the materials you need when using the Lonza FlashGelTM System. Jena Bioscience Direct PCR Lyophilised Master Mix. Please note, the protocol described here is for a 50 μl PCR reaction volume. The standard volume supplied by Jena Bioscience is 20 μl; but customised kits with a different reaction volume (such as 50 μl) can be ordered.

  • Optional: P1000 pipette tip to cut plant tissue
  • PCR racks
  • Thermal cycler
  • Jena Bioscience Direct PCR Lyophilised Master Mix
    (The kit contains DNA extraction buffer and PCR reagents (lyophilised dNTPs, DNA polymerase, buffers, stabilisers and BSA in PCR tubes, as well as an essential “additives mix“ in solution and RNase-free water.)
  • Optional: micro-pestles, sterile (for harder material such as plant seeds)
  • PCR primers to amplify rbcL and matK genes:
    • rbcL primers (working stock: 10 μM):
      Forward: Plant_Fwd_rbcLaf-M13 TGTAAAACGACGGCCAGTATGTCACCACAAACAGAGACTAAAGC
      Reverse: Plant_Rev_rbcLa-M13 CAGGAAACAGCTATGACGTAAAATCAAGTCCACCRCG
    • matK primers (working stock: 10 μM):
      Forward: matK-1RKIM Fwd-M13 TGTAAAACGACGGCCAGTACCCAGTCCATCTGGAAATCTTGGTTC
      Reverse: matK-3FKIM Rev-M13 CAGGAAACAGCTATGACCGTACAGTACTTTTGTGTTTACGAG

More information on the primers can be found here.

Agarose gel electrophoresis

This describes the materials you need when using the Lonza FlashGelTM System. Alternatively, you can use reagents/equipment for standard agarose gel electrophoresis. Note, in this case you may need to load a higher volume of the PCR product.

  • General electrophoresis power pack
  • Lonza FlashGelTM system: dock, camera, power supply and computer software
  • Lonza FlashGelTM gel(s)
  • Lonza FlashGelTM loading dye
  • Lonza FlashGelTM DNA marker (e.g. 50 – 1500 bp marker range)

Spin-column protocol

General equipment

  • Micropipettes (for a range of 1 to 675 μl; ideally P2, P20, P200, P1000)
  • Micropipette tips, sterile
  • 1.5 ml Eppendorf tubes, sterile
  • Vortex mixer
  • Centrifuge, speed-adjustable (20,000 x g and 6,000 x g)
  • Bench waste containers for small waste such as tips
  • Ice buckets and ice
  • Scissors
  • Permanent marker pens

DNA extraction

  • DNA extraction kit
  • Micro-pestles, sterile
  • Falcon tubes
  • Weighing boats
  • 65°C water bath or hot block

PCR

  • PCR reagents (e.g. PCR beads)
  • Thermal cycler
  • Sterile, RNase-free water
  • Degenerate primers (working stock: 10 μM):
    • Forward: Plant_Fwd_rbcLaf-M13* TGTAAAACGACGGCCAGTATGTCACCACAAACAGAGACTAAAGC
    • Reverse: Plant_Rev_rbcLa-M13* CAGGAAACAGCTATGACGTAAAATCAAGTCCACCRCG

* The primers can be sequenced using standard M13 forward and reverse sequencing primers. In the degenerate reverse primer the „R“ represents any purine (i.e. either adenine or guanine)

More information on the primers can be found here.

Agarose gel electrophoresis

  • Standard gel electrophoresis system
  • General electrophoresis power pack
  • Loading dye
  • DNA marker (e.g. 100 – 4k bp marker)

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