PCR on phage

Allison Hanly a.hanly at cheque.uq.oz.au
Sat Mar 12 03:02:44 EST 1994


On Wed, 9 Mar 1994 14:19:13 GMT, 
Gustavo Glusman  <bmgustav at dapsas1.weizmann.ac.il> wrote:

>Is there anybody who could give me some advice on how to run PCR directly on
>the supernatant contains phage particle?
>

I'm not sure about bacteriophage, but I use the following protocol to run PCR
on baculovirus in supernatant containg the virus

Adapted from Malitschek, B.M. & Schartl, M (1991) Biotechniques 11(2) 177-178

- Add 90 ul of detergent buffer to 10 ul of supernatant
 
  detergent buffer: 50 mM     KCl
                    10mM      Tris-HCl
                    0.1mg/ml  Gelatin
                    0.45%     NP-40
                    0.45%     Tween 20

- Add 6 ug Proteinase K
- Incubate 1 hr @ 60 deg.C
- Heat 10 min @ 95 deg. to inactivate the proteinase K
- Use 15 - 30 ul in a standard 50 ul PCR reaction 

- I use 30 - 40 cycles in the PCR reaction
- I can detect positive recombinants when I run 10 ul of the reaction on an 
  agarose gel.
- The reaction relies on having a high enough concentration of virus in 
  the supernatant (I can pick up recombinants at 10E4 to 10E5 pfu/ml) 

Hope this is of some use

Allison



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