isolation of DNA from mouse tails

Matthew P. Frosch, M.D., Ph.D. frosch at dsg.harvard.edu
Mon Jul 22 03:39:31 EST 1996


I have been using an extremely robust protocol for making tail DNA for
PCR.  THe original reference is: Laird, et al., NAR, 19:4293 (1991). 
Hope this helps.

Matthew Frosch.

Reagents:
	Tail digest buffer
		100 mM Tris-HCl, pH 8.5
		5 mM EDTA
		0.2% SDS
		200 mM NaCl
		100 mg/mL proteinase K (BMB)

	Isopropanol

	70% EtOH

	Sterile TE


SNIPPING THE TAILS

1.  Etherize mouse in appropriate facility
2.  Place ear tag on right ear.
3.  With sterile blade, cut approximately 1 cm off the end of the tail
and place in labeled eppendorf tube.
4.  Cauterize the wound to stop bleeding
5.  Return animal to clean cage.


DIGESTING THE TAILS

1.  Add 0.5 mL of tail; buffer with proteinase K to each tube.
2.  Digest overnight with rapid shaking at 55°C (tubes at 45° angle)
3.  Vortex vigourously.
4.  Spin in microfuge for 10 minutes.
5.  Pour supernatant into clean, labeled tube.


ISOLATING THE DNA

1.  Add 0.5 mL of isopropanol to each tube (work in groups of 6-8
samples)
2.  Invert until mixed
3.  Spin out DNA with P-200 pipette tip.
4.  Transfer DNA to labeled tube containing 0.5 mL of 70% EtOH
5.  Pellet DNA with a 1 minute spin.
6.  Aspirate 70% EtOH.
7.  Dry spin the tubes and remove residual EtOH
8.  Allow to air dry.
9.  Dissolve in 100 mL of TE with shaking at 55°C overnight.
10. Boil for 10 minutes
11. Spin hard for 5 minutes.
10. Store at 4°C.



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