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:
>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.
More information about the Methods