PCR Polishing Kit INSTRUCTION MANUAL
The Agilent PCR Polishing Kit is designed to increase blunt-ended cloning efficiencies associated with polymerase chain reaction (PCR)-generated fragments. The kit contains Cloned Pfu DNA polymerase, buffer, control DNA, and dNTP mix, allowing users to polish the ends of 3'-overhang extensions or perform complete fill-in of 5' overhangs directly from a PCR amplification reaction.
The manual details the materials provided, storage conditions at -20°C, and required additional reagents. It includes step-by-step protocols for the PCR Polishing Reaction, including specific volumes for control and sample reactions, incubation parameters (72°C for 30 minutes), and optional purification methods using phenol-chloroform extraction or the StrataPrep PCR purification kit.
| Reactions per kit | 40 reactions |
|---|---|
| Cloned Pfu DNA polymerase activity | 100 U (2.5 U/ μl) |
| Control DNA volume | 50 μl (10 ng/ μl) |
| dNTP mix concentration | 2.5 mM each |
| Incubation temperature | 72°C for 30 minutes |
The Cloned Pfu DNA Polymerase, Buffer, Control DNA, and dNTP mix should all be stored at -20°C.
Yes, they can be added directly to ligation reactions without further purification because Pfu DNA polymerase has very little activity at 25°C.
Transformation plates containing PCR-polished control DNA should exhibit a >20-fold increase in colony forming units compared to unpolished control DNA.