Last Updated: 02/12/16: 7:27 AM
Fast Genomic DNA Purification
Day 1:
Solutions:
Tail Buffer
50 mM Tris-HCl, pH8
100 mM EDTA, pH8
100 NaCl
1% SDS
for 25ml
1.25 ml 1M Tris-HCl, pH8
5ml 0.5M EDTA, pH8
0.5ml 5M NaCl
1.25ml 20% SDS
17 ml dH2O
Proteinase K
10 mg/ml in dH2O
1.
2.
Cut tails (about 1.5 cm) and place in 700l of Tail Buffer solution.
Add 35l of 10 mg/ml proteinase K and digest overnight at 50°C with agitation
(bottom of incubator will fit nutator).
Day 2.
Materials Needed:
100X RNAse
Isopropanol
70% EtOH
100% EtOH
TE pH8.0
Glass capillary tubes with one end sealed – 1 per sample and marked.
1.
Add 7l of 100X (10 mg/ml) RNAse and incubate at 37°C for 1-2 hours
2.
Centrifuge samples at high speed for 20 minutes to pellet hair and undigested bits
3.
Transfer supernatant to a fresh tube.
4.
Add 0.5ml isopropanol to tube and mix by rocking.
5.
Spool DNA onto sealed end of glass capillary tube. Spool along side of tube to
push out accumulated solution. Dip capillary tube in and out of 70% EtOH
solution multiple times (approx 10) and then repeat in the 100% EtOH solution.
6.
Air dry 30-60 minutes (10-15 min should be enough for them to dry)
7.
Break off glass containing DNA into 500l of TE.
8.
Nutate overnight at room temperature (can dissolve faster by placing at 50°C for a
couple of hours). Vortex every once in a while.
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Last Updated: 02/12/16: 7:27 AM
Day 3
1.
2.
3.
4.
5.
Vortex the samples and then remove the glass fragment.
If DNA is not in solution tubes can be placed at 50°C to help dissolve.
Read OD to determine the DNA concentration in each tube.
Dilute DNA samples 1:100 with water
(OD260)*(dilution)*(Constant)= X g/ml
Constants: DNA = 50 and RNA = 40
Do not need to check OD for PCr, only for Southern Blot
Use approx 4 l for PCr and 7 g for Southern Blot.
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