General Guidelines：
Reagent concentrations - "less
is usually better" (more specific)
primers: final concentration 0.1-1.0
uM
MgCl2: final
concentration 1.0-4.0 mM
(depends on Taq used)
dNTPs: final concentration 0.2 mM each dNTP (depends on Taq used)
note: sensitive to repeated
freeze/thaws
Vortex or finger-flick reagents to
mix well before use
Primer design very important
the higher the annealing temperature
the better (TANN > 50°C )
primer pairs should have melting
temps within 5°C of each
other
Annealing temperature and step times
are important
Titrations are a good idea
most commonly temperature and MgCl2
reactions often borderline resulting
in inconsistent amplifications
use Eppendorf Mastercycler Gradient
for titrations
titration
on Mastercycler Gradient
sample
titration gel
Hot starts improve reaction
efficiency (fewer primer-dimers)
manual: add Taq to tubes in
thermalcycler at 94oC (or MgCl2 or dNTPs)
TaqStart Antibody
Faststart Taq
hotstart
comparison gel
Always check program on
thermalcycler
Run negative control(s) to check for
contamination
Make a flow chart of what tried and
in what order
Run a positive control (a sample
known to amplify well)
Always run a ladder on gel (will
indicate whether failed PCR or failed detection system)
Additives for fragments that are
very long, G-C rich or prone to secondary structure
glycerol, formamide, NMP - lower
denaturing and annealing temperatures by a few degrees
DMSO decreases incidence of
secondary structure
For more tips see: http://www.biowire.com/nucleus/nucleus_1_1.jsp
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