Highly Convenient Tissue Digestion For Genotyping

April 06, 2018

University of Rochester Medical Center
Microbiology & Immunology
Research Scientist

Overall

Quality of Results

Ease-of-Optimization

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Company:

Vivagen

Product Name:

DirectPCR (Tail)

Catalog Number:

102-T

To process mouse tissue sample for genotyping, I had been using a classical method including DNA precipitation. However, once I used this direct lysis method from Vivagen, I no longer depend on the DNA precipitation method which takes long time and requires much more attention. Direct use of lysate never interfere with the PCR and rather guarantees more consistency.

Experimental Design and Results Summary

Application

PCR, genotyping

Starting Material

Mouse tail

Protocol Overview

Incubate cut mouse pup tail (usually 3-4mm- long) in less than 100 ul of the reagent at 55C overnight. The digestion reagent should be supplemented with proteinase K (usually 1 ul of most vendors such as NEB or Qiagen. One ul of proteinase K from NEB corresponds to 800mU). Vortex the tube containing the digest vigorously for 2-5 sec on next day (if you find a lot of liquid on the inside of the tube lid, take it down to the bottom either by gently and briefly spinning and vortexing again or by gently knocking the tube against the table). Then heat the tube at 85C for 45 min to inactivate residual proteinase K. Chill the digest to room temperature and vortex samples again to homogenize. Spin the tube just 1 min at 10,000-15,000g. It is ready for PCR now (the samples can be stored at 4C for a long time). Try to add the lysate less than 5% of the volume of PCR.

Tips

Do not use too large volume of the reagent; this will dilute the lysate to much. Keep the digest fully hydrated during the entire period of incubation; dried lysate may be incompletely digested and hard to rehydrate.

Results Summary

This lysate is completely compatible for regular PCR especially genotyping. I have never failed with this reagent. The attached figure shows a result to distinguish WT (lane 4 and 7) and red fluorophore protein-expressing transgenic (lane 1-3, 5,6) mice (first lane contained DNA standards).

DOI or PMID #

N/A

Additional Notes

N/A

Image Gallery

Summary

The Good

Extremely convenient compared to the classical method containing a DNA precipitation step

The Bad

Nothing

The Bottom Line

As long as you know a couple of things to be careful about in sample preparation with this reagent, you will never fail. This is super-convenient and I highly recommend.

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