The following is a DNA extraction protocol that i have found online. Does anyone know the ratio of phenol/chloroform that should be used?
C. ELEGANS GENOMIC DNA PREPARATION
Materials and Reagents:
Plates: NGM plates overlaid with 1% agarose—agar contains material that can inhibit
subsequent enzymatic manipulations of DNA
Lysis Buffer: 200mM NaCl, 100mM Tris-HCl pH 8.5, 50mM EDTA, 0.5% SDS
-aliquots are usually kept at -20°C
-add Proteinase K to 0.1mg/mL before use
3M Sodium acetate
- Wash worms from plate (NGM-AGAROSE) in dH2O;
this can be done in any size tube, but it is convenient to split worms so that
subsequent steps can be done in eppendorf tubes.
- Pellet by spinning at 3000rpm for 2min.
- Remove supernatant and add 5 volumes of lysis buffer with Proteinase K
(~100mL pellet of worms is convenient).
- Incubate at 65°C for 1-2hrs.
- Incubate at 95°C for 20-30min.
- Add DNAse-free RNAse to 0.1mg/mL and incubate at 37°C 1 hour.
- Extract 3 times with 1 volume phenol/chloroform.
- Add 0.1 volume 3M sodium acetate and >2 volumes 100% ethanol.
- Mix and incubate at RT or in the freezer for at least 1hr.
- Pellet DNA by centrifugation at 14000rpm for 15min.
- Remove supernatant and wash pellet with 70% ethanol.
- Air dry and resuspend in dH2O or TE.
- 3X phenol extraction should be sufficient, but if white precipitated material is
still visible at aqueous/organic interface, repeat extractions until it is no longer
- This prep is typically clean enough for PCR or restriction digests. If DNA is
to be used for injection into animals, you may wish to clean up DNA over a
spin column after restriction digest.
- Some protocols call for a freeze/thaw step to be included before lysis, but this
does not seem to be necessary.
Reference: Modified from Silverman lab protocol
Submitted by: James Morley 5/03
High School Senior