PCR of blood, hair or small tissue samples
Preparation of blood or a tissue sample obtained by ear punch for PCR
A single drop of blood, which can be obtained from mice at any age, should be added to 20 ul of the following mixture, and then 1 ul of 20 mg / ml Proteinase K added to the total:
50 mM Tris-HCl, pH 8.0 20 mM NaCl 1 mM EDTA 1% SDS This mixture should then be incubated at 55oC for 15 minutes, and then vortexed vigorously and incubated for a further 15 minutes at 55oC. Distilled water is added to bring the final volume to 200 ul. The sample is heated in boiling water for 5 minutes. After cooling, 1 ul of this sample may be used for PCR. Note: It has been reported that PCR of blood is NOT possible when the blood component of the reaction mixture exceeds 1%.
Ref. "A Simple Screening Method for Transgenic Mice Using the Polymerase Chain Reaction"; Shizhong Chen and Glen A. Evans, Biotechniques, Vol. 8, pp32-33 (1990).
Preparation of hair shafts for PCR
Microwave irradiation has been demonstrated to be effective as a pretreatment for the DNA extraction from fungi, plants and protists, and can be used in template preparation of both hair shafts and whole blood. 1 mg of hair or 5 ul of blood are microwaved (500W oven) for 4 minutes in microcentrifuge tubes with tightly fitting caps. After irradiation, the samples are chilled on ice and 50 ul of reaction mixture (below) added, and the samples cycled for 40 cycles of 30 seconds at 94oC, 90 seconds at 55oC and 60 seconds at 72oC, with a final extension at 72oC for 10 minutes. After amplification, 10 ul of PCR product can be visualized on a gel.
10 mM Tris-HCl, pH 9.0 50 mM KCL 1.5 mM MgCl2C 0.2 mM dNTPs 0.2 mM conc. of each primer 1% Triton X-100 0.05 U / ul Taq DNA polymerase
Ref. "Direct PCR of Whole Blood and Hair Shafts by Microwave Treatment"; Moritaka Ohhara, Yasuhiko Kirosu and Mariko Esumi, Biotechniques, Vol. 17, pp726-728 (1994).
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