Direct PCR protocol for bird feathers using Phire® Hot Start DNA Polymerase
* Cut a 1-2 mm piece of the quill tip, and place it directly into a 20 µl PCR reaction.
* Use the recommended protocol for Phire Hot Start DNA Polymerase, but increase the initial denaturation time at 98°C to 5 minutes and use a minimum of 20 seconds in the extension step. Use 40 cycles.
- 98°C 5 min
- 98°C 5 s
- X°C 5 s (depending on the primers; see instructions below and the product manual)
- 72°C 20 s for <1kb or 20 s/kb for amplicons longer than 1 kb
- 40 cycles
- 72°C 1 min
- 4°C hold
* The optimal annealing temperature depends on the primers. With Phire Hot Start DNA Polymerase, the optimal annealing temperature is typically over 60°C, but some primers may require lower temperatures. Temperature gradient is a useful tool in finding an optimal annealing temperature. It is recommended to use Finnzymes’ Tm calculator to set up the protocol.
* After PCR, spin down the reactions and take the supernatant to gel electrophoresis.
* The tip of the quill is the best starting material, most probably because it contains trace amounts of blood and other tissue. In some cases the rachis and/or barbs have also given positive results in direct PCR, but they seem to give more inconsistent results. We therefore strongly recommend using the quill tip.
* As in all direct PCR applications, it is highly recommended to include a positive control (purified DNA) to ensure that the PCR reaction conditions are optimal. A negative control without DNA template is also recommended.
* This protocol may require further optimization depending on the starting material. It is recommended to use as small a piece of feather as possible.
* These protocols are validated for PCR products up to 400 bp. Longer amplicons may require protocol optimization.
|F-120S||Phire Hot Start DNA Polymerase||200 rxn|
|F-120L||Phire Hot Start DNA Polymerase||1000 rxn|