Hirt Supernatents for Cloning Viral DNA

Dennis J. Templeton djt2 at po.CWRU.Edu
Wed Jan 29 13:25:21 EST 1992


In a previous article, bhjelle at vaxine.unm.edu (Brian Hjelle) says:

(stuff deleted)
>
>Does anyone have a protocol for Hirt supernatent preps that
>they would recommend? The only paper I have that talks about
>this in any detail is the one from Hirt himself (think he
>was trying to study T. rex viruses :-) ).

Ouch!  Ol' Bernard wouldn't think that funny.

Here is my Hirt prep from Grad school (used for Polyoma virus)

One 9 cm plate of infected cells, rinsed with pbs, at RT

Add 1.2 ml of (10 mM Tris 7.5 10 mM EDTA 0.6% SDS) 3-5 mins RT 
(rock to distribute)

Add 0.3 ml 5M NaCl, carefully scrape into microfuge tube.

Leave on ice 4 hours to overnight. 

Spin 15 k rpm, 15 mins

Pool all tubes together; Phenol Extract 1x; Phenol CHCl3 1x, CHCl3:IAA 1x

Ethanol PPT (2 vols, no added salt!) as usual.

The pellet will have lots of RNA, some broken genomic DNA and episomal DNA.

Good Luck,

dennis



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