Bio-Rad Fish DNA Barcoding Kit - Manual (Page 5)

Fish DNA Barcoding Quick Guide 3
8. Transfer the entire supernatant (500–550 µl)
of each fish sample from step 5 into the
appropriately labeled spin column. Try not to
get any of the particulates into the spin column
because they will clog the column and prevent
you from continuing.
9. Thoroughly mix the tube labeled Matrix by
vortexing or repeatedly shaking and inverting the
tube to make sure particulates are completely
resuspended before use.
10. Add 200 µl of thoroughly resuspended Matrix to
the first column containing fish extract and pipet
up and down to mix.
11. Using a new pipet tip, add 200 µl of thoroughly
resuspended Matrix to the second column
containing fish extract and pipet up and down to
mix.
12. Centrifuge the columns for 30 sec at full speed.
Take care to spin the column for only
30 sec. Drying the matrix completely at
this point will result in loss of DNA.
13. Remove the spin column from the 2 ml
microcentrifuge tube, discard the flowthrough
at the bottom of the 2 ml tube, and replace the
spin column in the same tube. Add 500 µl of
Wash and wash the matrix by centrifugation for
30 sec.
Take care to spin the column for only
30 sec. Drying the matrix completely at
this point will result in loss of DNA.
14. Repeat step 13 to wash samples again.
1 1
Supernatant
2 2
Supernatant
1
200 µl
Matrix
2
200 µl
Matrix
1 2
14,000 x g
30 sec
1
500 µl
Wash
2
14,000 x g
30 sec