Lambda gt11 insert PCR
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
> 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.
> 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
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.
email: foote at biologysx.lan.nrc.ca
"Nothing in science is fast or simple"
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