Suggestions needed for PCR

[Ed Rybicki]

> To:            methods-and-reagents at net.bio.net
> From:          rcuevas at umaxc.weeg.uiowa.edu (Ramon 'Jammin' Cuevas)
> Subject:       Suggestions needed for PCR
> Date:          22 Jun 93 03:05:42 GMT

> problems trying to amplify a much larger-sized insert. My question is this:
> can anybody tell me what "variables" can be modified that can possibly
> improve amplification (ie. changing the amounts of certain reagents; adding
> "catalytic" reagents; changing the number of cycles). In case this information
> is needed, the "recipe" I've had much personal success with includes the
> following:
>         1 (x2) uL of each primer
>         0.5 uL of Amplitaq (DNA polymerase) enzyme
>         5 uL of PCR buffer
>         8 uL of dNTP solution
>         1.5 uL of MgCl2
>         2 uL of DNA template
>         32 uL of water
>
>         94oC for 45 sec
>         45oC for 45 sec
>         72oC for 2 min

Yes, comrade - but WHAT CONCENTRATIONS of said reagents???  What are the
FINAL CONCENTRATIONS of your :
a) primers
b) dNTPs
c) MgCl2 ?

These matter - as do the TIMES for the thermal cycling: conventional
wisdom has it that one needs a little longer for denaturation for longer
templates, though obviously annealing will not be affected, and that you
need about 1 min at 72degC per kb of product (I use 1 min / kb) - so you
may need to change your thermal cycling profile.  You may also find, as
many have, that addition of DMSO to a max. of 10% helps production of
larger fragments: helps in denaturation.

These and other parameters are ably and amply discussed in:

Innis MA and Gelfand DH (1990).  Optimization of PCRs.  pp. 3-12 in: "PCR
    Protocols (Innis, Gelfand, Sninsky and White, eds.); Academic Press,
    New York.

    Good luck.

  ____________________________________________________________________
 | Ed Rybicki, PhD             |       "Lord, won't you buy me        |
 | (ed at micro.uct.ac.za)        |                                      |
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