Direct PCR from mouse ear and tail tissue samples

Three amplicons (194-466 bp) were amplified from a 0.5 mm ear or tail punch in a 50 µl reaction with a 2-step protocol (40 cycles). 7 µl of PCR products were run on FlashGel® System (Lonza) with DNARelease™ Additive in the FlashGel® Loading Dye. Overall protocol time from tissue to gel image was under 45 minutes. M, Size Marker. Piko® Thermal Cycler and UTW® reaction vessels were used in PCR.
 
Complete pipetting and cycling instructions can be found in the application note " Direct PCR from mouse tissue using Phire™ Hot Start DNA Polymerase".
 

Dilution PCR from mouse ear and tail samples

A 2 mm ear punch or 1 mm tail section were placed in 20 µl of TE buffer containing 0.5 µl of DNARelease Additive. After vortexing for 15 seconds, the samples were incubated at 75°C for 5 minutes and then at 96°C for 2 minutes. After centrifugation, 0.5 µl of supernatant was used as a template in a 5 µl PCR reaction. Reactions were run on Piko® Thermal Cycler using UTW® reaction vessels and a 2-step PCR protocol (35 cycles). The PCR products were separated and visualized on FlashGel System® (Lonza). The overall protocol time was under 30 minutes. M, Size Marker.

 

General notes

  • 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.
  • Mouse ear tissue often gives better results than mouse tail tissue.
  • These starting materials have been tested only with end point PCR protocols.