Lambda gt11 insert PCR

Simon Foote foote at biology.lan.nrc.ca
Thu Jan 5 11:19:23 EST 1995


In article <1995Jan4.145054.1 at icbr2.ifas.ufl.edu>, aris at icbr2.ifas.ufl.edu
wrote:

> Dear Colleagues,
> 
>         We are interested in using PCR to amplify inserts from lambda
> gt11, and are familiar with doing this using purified lambda DNA as
> template.  Can inserts be amplified without purifying the lambda DNA?
> If so, and suspensions of lambda phage particles in SM buffer can be
> used in PCR, how is this done?  How much plate or liquid lysate is
> needed?  Is a published method(s) available?  Related comments and 
> anecdotes would be appreciated.
> 
> Thanks,
> 
> John Aris
> aris at icbr.ifas.ufl.edu

The inserts can be effectively amplified without DNA purification.  The
method I have used that worked well is:


Grow an o/n of your phage stock with shaking
Next day, add 20ul chloroform and shake 15min 37oC
Pellet debris
Take 1ml of supernatant and add 10ul chloroform
This can be stored at 4oC

For PCR, use 1ul of this stock solution as your template.

I haven't tried it straight from a plate lysis in SM buffer, but it may
work.  I'd use a couple microlitres as template and see what happens.


Simon Foote
Novel Antibodies
NRC
email:  foote at biologysx.lan.nrc.ca
[Usual disclaimers]
"Nothing in science is fast or simple"



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