Protocol
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PCR Product Clean-Up
Prior to Sequencing
The clean-up reaction removes unincorporated primers
and degrades unincorporated nucleotides. The resulting
PCR product is ready to use for sequencing without
additional purication, e.g. using column purication kits.
1. Prepare the following reaction mixture:
PCR mixture (directly after completion of PCR) 5 µl
Exonuclease I (Exo I) 0.5 µl
(10 u)
FastAP™ Thermosensitive Alkaline Phosphatase
or Shrimp Alkaline Phosphatase (SAP) 1 µl (1 u)
2. Mix well and incubate at 37°C for 15 min.
3. Stop the reaction by heating the mixture at 85°C for
15 min.
Note
• Up to 5 µl of puried PCR products can be used directly
for DNA sequencing without further purication.
• For reliable sequencing results there should not be
non-specic PCR products.
• The protocol may be applied for clean-up of PCR
products, generated by any thermophilic DNA poly-
merase or polymerase mix.
• The procedure is not recommended for downstream
cloning applications.
Reference
1. Werle, E., et al., Convenient single-step, one tube
purication of PCR products for direct sequencing,
Nucleic Acids Res., 22, 4354-4355, 1994.
This protocol is for the PCR Product Clean-Up Prior to Sequencing
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