CMarZ Barcoding Association
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 C6, 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.