Introduction

This protocol describes a phenol-chloroform based method to purify high molecular weight genomic DNA embedded and stored in a 1%, low-melting point agarose plug. We tested this protocol using both Lambda DNA that we embedded in 1%, low-melting point agarose and S. cerevisiae DNA embedded in 1%, low-melting point agarose.

Materials

• 1 x agarose plug
• 2 ml Eppendorf tubes
• Incubator, water bath or equivalent (set to 70°C)
• TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0)
• NaCl 5 M
• Phenol
• Chloroform
• Centrifuge (capable of 9400 x g)
• HulaMixer or equivalent
• Ethanol
• Ammonium acetate 5 M
• Freezer (-20°C)

Methods

1. Transfer an agarose plug (1% low-melt point) containing ~3-6 µg of DNA, to a 2 ml Eppendorf tube and add 200-400 µl TE to cover the agarose. If the agarose is on the tube wall, briefly centrifuge the tube. Add NaCl to a final concentration of ~200 mM.

2. Melt the agarose at 70°C. The solution will become transparent and homogeneous, which will take approximately 5 minutes.

3. In a fume hood, add 1 volume of phenol and gently rotate in a HulaMixer for 2 hours at room temperature.

4. Centrifuge the tube for 5 minutes at 9400 x g.

5. In a fume hood, retain and transfer the supernatant to a new 2 ml tube and add 1x volume of chloroform.

6. Thoroughly but gently invert to mix. We recommend ~25 inversions.

7. Centrifuge the tube for 5 minutes at 9400 x g.

8. In a fume hood, retain and transfer the supernatant to a new 2 ml tube and add 2.5x volumes of 100% ethanol and 1/100 volume of 5 M ammonium acetate.

9. Invert 10 times and incubate overnight at -20°C.

10. Centrifuge the tube for 5 minutes at 9400 x g.

11. Discard the supernatant and retain the pellet.

12. Add 1 ml of ice-cold 70% ethanol and invert.

13. Centrifuge the tube for 5 minutes at 9400 x g.

14. Repeat Steps 11-13.

15. Discard the supernatant and retain the pellet. Allow the pellet to air-dry for 1 minute.

16. Resuspend the pellet in 25 µl TE and incubate at room temperature for 2 hours.

Lambda DNA

Results

• Yield: 50-80% of initial DNA amount
• OD 260/280: 1.88
• OD 260/230: 1.79

Sequencing performance

Libraries were prepared using the Ligation Sequencing Kit.

• Read length profile:

S. cerevisiae DNA

Results

• Yield: 50-80% of initial DNA amount
• OD 260/280: 2.11
• OD 260/230: 1.85

Sequencing performance

Libraries were prepared using the Ligation Sequencing Kit.

• Read length profile:

Change log

Version	Change
v1, June 2019	Initial protocol publication