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
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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.
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| 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 |
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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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Allowed tags: i, b, u, sup, sub 
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| | 06/21/2008 3:48:55 PM
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shivali responded with a statement of type: Neutral
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why did the negative control also showed amplification...
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| | 06/26/2008 4:44:12 AM
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Linda responded with a statement of type: Neutral
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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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| | 08/31/2010 4:10:12 PM
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SIAL SAW responded with a statement of type: Agree
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Linda is correct here. If your primers are not the best primers you could use in your PCR reactions you will get non-specific products and/or primer dimer. If you ran these reactions out on a agarose gel I would expect to see a fair amount of primer dimer. You can excluded this dimer product from your SYBR Green qPCR final Ct by putting specific melt-read points in the analysis.
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| | 12/02/2008 11:16:24 PM
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Thierry responded with a statement of type: Agree
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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
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Padma Sudarshana responded with a statement of type: Neutral
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Does Extract-N-Amp work for gram positive bacteria? I just have to get amplifiable DNA from gram positive bacteria
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| | 08/31/2010 4:35:00 PM
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SIAL SAW responded with a statement of type: Neutral
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We have not developed a protocol for Gram-Positive Bacteria currently.
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| | 03/25/2009 5:36:39 PM
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wuxing Dong responded with a statement of type: Question
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How much of each PCR product should be loaded to gel for separation?
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| | 08/31/2010 4:17:43 PM
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SIAL SAW responded with a statement of type: Neutral
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I have always loaded 5 ul of the RED PCR product. With the specific kit mentioned in the above video, the kit has a "clear" 2X PCR mix provided, which can still be loaded on an agarose gel, but I would add 4 ul of a loading buffer, such as Sigma's G2526 to the whole PCR reaction, then load 5 ul on the gel.
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| | 08/12/2009 2:46:01 PM
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aaaaaaaa responded with a statement of type: Neutral
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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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| | 08/31/2010 4:25:20 PM
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SIAL SAW responded with a statement of type: Neutral
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Usually, I recommend that if you store the extract for more than 2 weeks at 4C, I take the mouse tail or tissue out. Same goes for long term storage at -20C, but I would recommend taking the mouse tail out before you freeze the extract. The mouse tail will start to degrade if kept on solution for long term. This action can release more PCR inhibitors into the extract. You should be able to take the extract out of long term storage and use it in PCR reaction again with positive results.
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| | 02/15/2010 3:17:23 PM
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Mike responded with a statement of type: Question
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Does anyone know post-digestion of product, what the precipitate is at the bottom?
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| | 08/31/2010 4:31:08 PM
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SIAL SAW responded with a statement of type: Neutral
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Depending on the starting material, you may get some precipitant in the bottom of the tube after the extraction protocol in done. Do not worry about it, if you are worried about it, a quick spin down of the extract before placing an aliquot in PCR will ensure that nothing is drawn up in your sampling.
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| | 08/05/2010 5:06:20 PM
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Anonymous responded with a statement of type: Neutral
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Is this discussion still alive?
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| | 08/31/2010 4:31:43 PM
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SIAL SAW responded with a statement of type: Neutral
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Yes
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