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CLONTECH Laboratories, Inc.

V. Troubleshooting Guide

The following general guidelines apply to most PCR reactions. However, no

attempt has been made to address troubleshooting for all of the many applica-

tions for which the Advantage-HF Kit can be used. When using the kit with

another CLONTECH product, additional, application-specific troubleshooting

information can be found in the relevant User Manual.

A. No product observed

PCR component

missing or degraded perform a positive control to ensure that each com-

Too few cycles

Annealing temp.

too high

Suboptimal primer

design

Not enough

template

Poor template

quality

Denaturation temp. Optimize denaturation temperature by decreasing or

too high or low

Denaturation time

too long or too short ing it in 10-sec increments. (A denaturation time that

Extension time too

short

page

Protocol # PT3139-1

Version # PR76834

Use a checklist when assembling reactions. Always

ponent is functional. If the positive control does not

work, repeat the positive control only. If the positive

control still does not work, repeat again replacing

individual components to identify the faulty reagent.

Increase the number of cycles (3–5 additional cycles

at a time).

Decrease the annealing temperature in increments

of 2–4°C.

Redesign your primer(s) after confirming the accu-

racy of the sequence information. If the original

primer(s) was less than 22 nt long, try using a longer

primer. If the original primer(s) had a GC content of

less than 45%, try to design a primer with a GC

content of 45–60%.

Repeat PCR using a higher concentration of DNA

(after trying more cycles).

Check template integrity by electrophoresis on a

standard TBE-agarose gel. If necessary, repurify

your template using methods that minimize shearing

and nicking.

increasing it in 1°C increments. (A denaturation

temperature that is too high can lead to degradation

of the template, especially for long target sequences.)

Optimize denaturation time by decreasing or increas-

is too long can lead to degradation of the template,

especially for long target sequences.)

(Especially with longer templates) Increase the

extension time in 1-min increments.

Technical Support TEL:415-424-8222 or 800-662-CLON

FAX: 415-424-1064 or 800-424-1350