Tech Tips tenplate temp 10/12/98 5:50 pm Page 5

Technical tips

Isolation of DNA from sperm using

Nucleon BACC 2
D. R. Marchington
Department of Paediatrics, John Radcliffe Hospital, Oxford, UK

Using an adaptation of the Nucleon™ BACC 2 protocol has

enabled the optimization of DNA yields from sperm samples
from infertile patients with probable low sperm counts.

Procedure
1. Resuspend 500 µl sperm sample in 2 ml 1× SSC and
centrifuge at 3,000 rpm for 2 min. Perform two further
washes using 2 ml 1× SSC, discarding the supernatant
each time.

2. Resuspend the pellet in 2 ml Reagent B (with 1 M β-

mercaptoethanol added), vortex and incubate at 37 °C
for 1 hr.

3. Add 500 µl sodium perchlorate solution and rotary mix

at room temperature for 15 min followed by 25 min on
a shaking platform in a 65 °C oven.
4. Add 2 ml chloroform (stored at -20 °C) and rotary mix
at room temperature for 10 min.

5. Centrifuge at 800 g for 1 min.

6. Add 300 µl Nucleon resin and centrifuge at 1,400 g for

3 min.

7. Carefully remove the supernatant and respin to remove

any residual resin.

8. Recover the supernatant and add two volumes of ice-

cold absolute ethanol, and invert the tube to mix.

9. Leave overnight at -20 °C and centrifuge at 1,400 g to

pellet the DNA.

10. Wash the pellet once using 70% ethanol and resuspend
in 250 µl TE buffer.

Yields of 50–135 µg DNA were obtained from 500 µl samples,

and 1 µl aliquots of the final DNA suspensions were used
successfully in PCR.

Life Science News 1, 1998 Amersham Biosciences