Abstract: The “Freeze squeeze” method is a cost effective and technically simple procedure to isolate and prepare PCR products for DNA sequencing.

Reagents

• 70% ethanol
• 3 M sodium acetate

Procedure

1. Run your PCR product of interest on a 1.5% agarose gel at 80V for 45 min.
2. Cut the band out of the agarose gel under UV light (be warned UV light will burn your naked skin, wear protective clothing and safety glasses.)
3. Place excised band into a 1.5 ml tube.
4. Freeze for a minimum of 30 min, 24 hours preferable.
5. Grab a section of parafilm (10cm by 6cm). Fold it in half, now with the fold being on the left side fold the bottom 2 cm back on itself. This should form a square pocket that is closed on two sides and open on two sides.
6. Place frozen gel block into pocket, so that its trapped in the closed right angle of the pocket.
7. Gently squish the gel block (be careful its slippery)
8. The resulting liquid is removed via pipette (a P20 is best) and placed into a new 1.5 ml tube.
9. Measure the amount of freeze squeeze supernatant collected.
10. Add one tenth the extraction volume of 3M Sodium Acetate and twice the extraction volume of 70% ethanol to the freeze squeeze liquid
11. Spin tube at 14000 rpm for 5 min
12. Discard the supernatant, be careful not to disturb the pellet at the bottom of the tube.
13. Dry the pellet in a speed vac for 15 min
14. Resuspended the pellet in 15 μl of PCR grade water or TE buffer
15. Run 2 μl of the resuspended DNA on an agarose gel to quantitate the amount of DNA present or use a nanodrop machine.
16. Your DNA is now clean and pure and ready to undergo a PCR sequencing reaction.

I would like to acknowledge Ms Linda McInnes (Molecular Epidemiology) and Miss Frances Briggs (SABC) of Murdoch University, Perth Australia for introducing me to this technique. Any question regarding this technique - DNATAQ@gmail.com.