Mouse Tail DNA Extraction

 

1. Cut 0.5 cm of tail and mark mouse ears or toes

 

2. Incubate at 55°C O/N in:

 

500 l Tail Extraction Buffer

10 l Proteinase K (10 mg/ml)

12.5 l 20% SDS

 

3. Add 150 l 5 M NaCl

Vortex 5 min

Microfuge 15 min, 14,000rpm (12,300 rpm on larger-radius centrifuge)

 

4. Label second set of eppendorfs

Add 1 ml of 100% EtOH

 

5. Transfer 500 l of supernatant to these tubes

Invert several times to precipitate DNA (looks like cotton wool)

Microfuge 10 min 14,000rpm (12,300 rpm)

 

6. Remove EtOH by suction - double tip and be careful not to lose the pellet

Resuspend DNA pellet in 200 l of TE by scraping on rack and vortexing 15 min

 

7. Reprecipitate DNA with 500 l 100% EtOH

Invert several times to precipitate DNA

[If DNA does not precipitate, add salt (0.3 M final concentration). Generally the samples contain excess NaCl which is sufficient to precipitate the DNA. If the salt is not removed, it will prevent restriction of DNA with endonucleases]

Microfuge 10 min 14,000 rpm (12,300 rpm)

 

8. Remove the EtOH completely

Air dry the DNA pellet for 5-15 min (not more)

Resuspend DNA in 200 l of TE by scraping on rack and vortexing 15 min.

 

9. Restriction Enzyme Digest

Premix: 4l Bam H1, 4 l 10X buffer, 0.4 l BSA /sample

Aliquot 8.4 l / third set of tubes

Add 31.6 l genomic DNA and pipet up and down to mix

Digest 5hrs-O/N at 37° C

 

10. Load onto an 0.8% agarose/TAE gel containing 0.5ug/ml ethidium bromide (10 l of 20mg/ml stock/200ml gel):

Minigel 50mls; 120V/hrs, 40V for 3hrs or 10V O/N

Maxigel 400mls: 360V/hrs, 30V O/N or 120V for 3hrs

(3.2g agarose in 400ml 1X TAE plus 20 l ethidium bromide)

 

Tail Extraction Buffer = 50 mM Tris-HCl (pH 7.4), 100 mM EDTA, 100 mM NaCl

1 M Tris-HCl (pH 7.4) 5 ml

0.5 M EDTA 20 ml

5M NaCl 2 ml

Qs. to dH2 0 100 ml

Autoclave