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.