CMarZ Barcoding Association

Protocols

Total DNA Extraction from small metazoans¹. Modified from Schizas et al. (1997)

1) Prepare 1x PCR Buffer
add 10x Buffer 1 µl ・・・Typical PCR Buffer
and DW 9 µl
2) Put 10 µl of prepared 1x PCR Buffer for each 0.2 ml tube.
3) Put specimens into the tube.
4) Heat the tube, 94C, 2 min, with thermal cycler². Return the on ice.
5) Add 1 µl of Protease K in each tube.
6) Incubate 55C, 15min followed by 70C, 10 min³. Return the on ice.
7) Add 10 µl of GeneReleaser⁴.
8) Follow thermal profile,
65C (15sec)→8C (15sec)→65C (45sec)→97C (90sec)→8C (30sec)→
　　　65C (90sec)→97C (30sec)→65C (30sec)→80C (3min)→4C (∞)
9) Centrifuge with max speed, 1min.
10) Transfer the supernatant into fresh tube (about 15 µl will be collected).
11) Add 10 µl of TE buffer into the supernatant⁵.
12) Store in -20 C^{6, 7}.

¹ This protocol is appropriate for animals with body sizes less than ~2mm.
² This step is not written in the original protocol, but will increase success of PCR amplification. The purpose of this step is to denaturize the protein.
³ In the original protocol, inactivation of Protease K is carried out at 100C. But 70C is enough with recent thermal cycler.
⁴ Mix well before use.
⁵ Addition of TE buffer will stabilize extracted DNA.
⁶ Extracted DNA with this protocol contains silica resin Glassmilk® from GeneReleaser, so measurement of DNA concentration with spectrophotometer is not possible.
⁷ Before using extracted DNA for PCR amplification, centrifuge briefly. Do not carry over contaminated Glassmilk for PCR solutions.