Agarose plug DNA
Extraction Method
Agarose plug DNA
FOR RESEARCH USE ONLY
Contents
Introduction
Materials
Methods
Lambda DNA
*S. cerevisiae* DNA
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
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.
Melt the agarose at 70°C. The solution will become transparent and homogeneous, which will take approximately 5 minutes.
In a fume hood, add 1 volume of phenol and gently rotate in a HulaMixer for 2 hours at room temperature.
Centrifuge the tube for 5 minutes at 9400 x g.
In a fume hood, retain and transfer the supernatant to a new 2 ml tube and add 1x volume of chloroform.
Thoroughly but gently invert to mix. We recommend ~25 inversions.
Centrifuge the tube for 5 minutes at 9400 x g.
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.
Invert 10 times and incubate overnight at -20°C.
Centrifuge the tube for 5 minutes at 9400 x g.
Discard the supernatant and retain the pellet.
Add 1 ml of ice-cold 70% ethanol and invert.
Centrifuge the tube for 5 minutes at 9400 x g.
Repeat Steps 11-13.
Discard the supernatant and retain the pellet. Allow the pellet to air-dry for 1 minute.
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 |