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Rapid Genotyping of Mouse Tissue Using Sigma's Extract-N-Amp Tissue PCR Kit
Linda Doan, Edwin S. Monuki
Department of Developmental and Cell Biology, University of California, Irvine

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0:00 Title0

0:12 Introduction12

1:04 Preparing Tissue and PCR Reaction Mixture64

6:58 Real Time Quantitative PCR418

11:10 Melting Curve Analysis670

11:45 Conclusion705

Genomic detection of DNA via PCR amplification and detection on an electrophoretic gel is a standard way that the genotype of a tissue sample is determined. Conventional preparation of tissues for PCR-ready DNA often take several hours to days, depending on the tissue sample. The genotype of the sample may thus be delayed for several days, which is not an option for many different types of experiments. Here we demonstrate the complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR Kit. First, we demonstrate the fifteen-minute extraction of DNA from the tissue sample. Then, we demonstrate the real time read-out of the PCR amplification of the sample, which allows for the identification of a positive sample as it is being amplified. Together, the rapid extraction and real-time readout allow for a prompt identification of genotype of a variety different types of tissues through the reliable method of PCR.

Please click here to view the protocol.

Name  Type  Company  Catalog Number  Comments
SYBR Green Extract-N-Amp Tissue PCR Kit Reagent Kit Sigma XNATG Includes: Extraction Solution Neutralization Solution B Tissue Preparation Solution Extract-N-Amp SYBR Green PCR ReadyMix
Doan L, Monuki ES (2008). Rapid Genotyping of Mouse Tissue Using Sigma's Extract-N-Amp Tissue PCR Kit. JoVE. 11. http://www.jove.com/index/details.stp?id=636, doi: 10.3791/636
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06/21/2008 3:48:55 PM
shivali responded with a statement of type: Neutral

why did the negative control also showed amplification...

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06/26/2008 4:44:12 AM
Linda responded with a statement of type: Neutral

It depends on how good the primers for the PCR are.  We used a mediocre primer, and it did have some non-specific amplification that came up later in the cycling, and was deemed negative.  Before we used the Extract-N'Amp on experimental tissue, we made sure our to test different positive samples which consistently amplified early in the cycling.

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06/21/2008 3:50:20 PM
shivali responded with a statement of type: Neutral

why did the negative control also showed amplification...

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12/02/2008 11:16:24 PM
Thierry responded with a statement of type: Agree

I'm using the same kit from Sigma to genotype KO mouse, it is highly reliable and works pretty well.

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01/29/2009 2:45:20 PM
Padma Sudarshana responded with a statement of type: Neutral

Does Extract-N-Amp work for gram positive bacteria? I just have to get amplifiable DNA from gram positive bacteria

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01/29/2009 2:46:55 PM
Padma Sudarshana responded with a statement of type: Neutral

Does Extract-N-Amp work with gram positive bacteria

 

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03/25/2009 5:36:39 PM
wuxing Dong responded with a statement of type: Question

How much of each PCR product should be loaded to gel for separation?

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08/12/2009 2:46:01 PM
aaaaaaaa responded with a statement of type: Neutral
How long time can DNA that extracted using redxtract kit store at -20C? I did same with you before, the dna can not PCR again after 6 months.
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01/22/2008

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doi: 10.3791/636 

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Journal of Visualized Experiments (JoVE) is an online research journal employing visualization to increase reproducibility and transparency in biological sciences.

 

ISSN 1940-087X

 

 

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