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Phire Animal Tissue Direct PCR Kit


PCR directly from various animal tissues including mice, fish, birds and insects without DNA purification.
  
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Animal IconThermo 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
Animal Protocol

Highlights

  • No need for time-consuming and expensive DNA purification steps
  • Very little sample material required
  • Two simple protocols for various applications
  • Convenient sampling tools included in the kit
  • Phire Hot Start II DNA Polymerase provides high yields of specific product with short extension time (20 s/kb).

Includes

  • Phire Hot Start II DNA Polymerase
  • 2X Phire Animal Tissue PCR Buffer (includes dNTPs and MgCl2)
  • Universal control primer mix
  • Dilution Buffer
  • DNARelease Additive
  • Gel loading dye
  • Harris Uni-Core™ 0.50mm
  • Harris Cutting Mat™

Two alternative protocols suitable for various purposes

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.

Recommended Sample Sizes

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).

Tissue sample Direct Protocol
(50 µL reaction)		 Dilution Protocol1
(20 µL Dilution Buffer + 0.5 µL DNARelease Additive)
C. elegans 2-25 worms 25-40 worms2
Drosophila Wing (e.g. 1 mm x 2 mm) Whole Drosophila
Mouse ear 0.5 mm punch 2 mm punch
tail 0.5 mm punch 1-3 mm tail section
embryo - 0.5 x 1 mm piece
organs (such as spleen)3 1 x 1 mm piece 1 x 1 mm piece
cultured cells (fibroblasts)4 - 104 cells
Zebrafish fins (tail) 0.5 mm punch 1 mm x 5 mm
Whitefish fins (tail) 0.5 mm punch 1 mm x 5 mm
scale 0.5 mm punch 1 scale (approx. 2 mm diameter)
Agrilus viridis - One leg or 0.5 x 1 mm piece of body
Animal hair 1-2 mm region with follicle 2-3 mm region with follicle
Bird feather 1-2 mm piece of quill end 2-3 mm piece of quill end
Muscle tissue 0.5 mm punch 2 mm punch
  1. Make sure the sample is covered with solution. If larger samples are used, adjust the volumes of Dilution Buffer and DNARelease Additive accordingly.
  2. If less worms are used, adjust the volume of the Dilution Buffer and DNARelease Additive accordingly.
  3. The direct protocol is not recommended for fat or bone tissue.
  4. The cells were trypsinized, washed with PBS and collected by centrifugation.

Recommended control primer sequences for zebrafish and Drosophila

Species Primer Sequence Melting point (Tm)
zebrafish (378 bp) Primer # 1 (20-mer)TAT AGC CCA GGG ACA CGA AC 64.17°C
zebrafish (378 bp) Primer # 2 (20-mer)CAG CTG CTC CAA AAA CAA CA 64.38°C
Drosophila (300 bp) Primer #1 (21-mer)AAG GGC AGT TTT GGT GGA GAT 66.05°C
Drosophila (300 bp) Primer #2 (21-mer)AGT GCG ATG CTT GAT GAA AAC 64.44°C

 

  
Storage Condition-20 C
HazardousNo
Phire Animal Tissue Direct PCR Kit yields robust genotyping results

Genotyping F2 transgenic mice

Phire Animal Tissue Direct PCR Kit yields robust genotyping results

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).


Phire Animal Tissue Direct PCR Kit amplifies from a variety of animal tissues.

Direct PCR from various animal tissues

Phire Animal Tissue Direct PCR Kit amplifies from a variety of animal tissues.

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.


Citations

  1. H. Yoshida, A. Nozawa, N. Kanda, T. Kishiro and T. Miyashita. Results of onboard genetic analysis of common minke whale biopsy samples collected in the Okhotsk Sea, summer 2010. Document SC/D10/NPM9 submitted to the first intersessional workshop for North Pacific Minke Whale Implementation Review, (2010).

  2. P. Bartonova et al., Association between CSN3 and BC02 gene polymorphisms and milk performance traits in Czech Fleckvieh cattle breed. Genet. Mol. Res. 11(2), 1058-1063 (2012). [DOI http://dx.doi.org/10.4238/2012.April.27.4.]