Posted on June 28th, 2016 by UNM CC
Lysis Buffer: (10 mM Tris pH8, 100 mM NaCl, 25 mM EDTA, 0.5%SDS), store at room temp.
10 mg/mL Proteinase K: 100 mg (Roche) + 10 mL buffer (10 mM Tris pH8.0, 20 mM CaCl2, 50% (v/v) glycerol), store at -20ºC in 1.5 mL aliquots.
Lysis buffer + proteinase K (1 mg/mL): 50 mL Lysis buffer (above) + 0.5 mL 10 mg/mL proteinase K, store at -20ºC.
TE: 10 mM Tris pH8, 1mM EDTA
Phenol: Molecular biology grade. Equillibrate 1x with Tris pH8, and then 1x with TE Store at 4°C.
Chloroform: Fresh choloroform + 1/20 vol. isoamyl alcohol. Store at r.t.
- Cut 3 mm of toe or tail or toe tips from 7-10 day old mice into 1.5 mL microcentrifuge tubes.
- Digest tail biopsy by adding 0.7 mL lysis buffer + proteinase K and incubate for 4-16 hrs at 37°C.
- Add 0.7 mL of phenol (or a 1:1 (v:v) mix of chloroform:phenol) and slowly rotate at 4°C for 1 hr. to overnight.
- Spin at high speed (15,000 g) for 2 minutes to separate phases. The hair and other debris should pellet to the bottom. Pipet the top (aqueous) phase to a fresh tube, taking care to avoid the bottom (organic) layer.
- Repeat the extraction (as in steps 2-3) with 0.7 mL of phenol or a 1:1 (v:v) mix of chloroform:phenol for 1 hr.
- Repeat the extraction (as in steps 2) but with 100% chloroform. You should now have ~0.7 mL of aqueous solution in each tube. Spin for 2 min. Transfer 0.5 mL the solution to a fresh tube, taking care not to place the pipet tip to the bottom of the tube (since this is where any residual CHCl3 will be located).
- Precipitate by adding 2 volumes (1.0 mL) of absolute ethanol and mix vigorously. You should see some DNA stranding at this point. Incubate at -20°C for >30 min. Samples may be stored in -20ºC in ethanol indefinitely.
- Spin at 12,000 rpm for 5 min. Discard supernatant. Wash pellet by adding 1 mL of 70% ethanol, mix by inversion, and spin for 1 minute. Discard supernatant being taking care not to pour out the pellet. It is likely to be loosely adherent since the salt is washed out. Repeat the 70% ethanol wash, spin and again discard the supernatant. Blot the excess ethanol carefully on a clean paper towel. Invert the tube on a paper towel to air dry the tube.
- Resuspend pellet in 0.1-0.5 mL TE (depending on the amount of DNA). Use 1 µL for a PCR reaction, or 1/2 of the sample for a Southern blot. Store the genomic DNA at 4ºC for 1-2 weeks, or at -20ºC for longer periods.
Quality Control - Options for QC include the following:
- Run 2 µL of the genomic DNA on a 0.6% agarose gel, with high MW marker, e.g. lambda HindIII. The DNA should form a high MW band (20-50 kb), with minimal smearing or degradation below this level. RNA may also be present if a metabolically active source tissue was used instead of toe/tail tip. Insoluable material, or protein contamination, may result in ethidium bromide staining that does not migrate out of the loading well.
- A spectrophotometer (e.g.Nanodrop) may also be used to measure the A250/A280 ratio, as a crude indicator of DNA purity. Values of 1.9-2.0 are expected. Values > 2.0 may indicate organic solvent contamination; values <1.9 represent protein contammination.
Quantitation - Options for quantifying genomic DNA include:
- The DNA concentration may be estimated by comparing band intensity on an agarose gel to lamda HindIII or other standards with known band quantitities.
- The spectrophotometric A260 value may be used to estimate DNA concentration, if the DNA is high quality. ([DNA] mg/mL = A260 x 50)
- Fluorometry with a DNA binding dye (SybrGreen or Höchst) is an accurate and sensitive measure of DNA concentration.