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On-demand webinars:Direct PCR Kits and Applications
Thermo Scientific Phire Animal Tissue Direct PCR Kit has been developed for amplification of DNA directly from a wide variety of animal tissues including mice, fish, birds and insects.The kit contains all the necessary components for amplification of DNA directly from animal tissues: optimized reagents, sampling tools, and DNARelease Additive, which can be used to improve the release of DNA from animal tissues. In addition, the kit includes detailed instructions and control primers that can be used with numerous animal species.
Animal Direct PCR Protocol
In the direct protocol, a small piece of animal tissue is placed directly into the PCR reaction. In the dilution protocol, a small amount of animal tissue is first briefly incubated in incubation buffer and 1 μL of this buffer is then added into the PCR reaction. The dilution protocol is useful if several PCR reactions are performed from the same sample.
The optimal annealing temperature for Phire DNA Polymerases may differ significantly from that of Taq-based polymerases.
For optimal results start by accurately calculating your Tm with our Tm calculator.
The sample size recommendations below are based on organisms and tissue types that have been tested so far. Some optimization may be needed when starting to perform Direct PCR reactions on a new sample, but the recommendations can be used as guidelines.
In general, it is beneficial to take as small a piece of the sample as possible to minimize the amount of inhibitors to the reaction.
Note on the Direct Protocol: The Direct Protocol is recommended only for short amplicons (<500bp for fish fins and < 1kb for other tissues).
TAT AGC CCA GGG ACA CGA AC
CAG CTG CTC CAA AAA CAA CA
AAG GGC AGT TTT GGT GGA GAT
AGT GCG ATG CTT GAT GAA AAC
0.50 mm punches of mouse ear tissue were placed directly into 50 µL PCR reactions. Two sets of primers were used in each reaction to genotype the 11 individual mice. The larger fragment was 490 bp (transgenic) and the smaller fragment 250 bp (wild type).
Small samples of various animal tissues were placed directly into 50 µL PCR reactions. A specific DNA fragment was amplified with control primers included in the kit or, in the case of fruit fly and zebrafish, using control primers whose sequences are provided at www.thermoscientific.com/directpcr. + and - denote control reactions with or without purified DNA.