Journal of Visualized Experiments

Sign In
New Account Forgot Password
Search
Home Browse For Authors Subscribe
< Previous Article
Next Article >
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

Sorry for the inconvenience.
This content requires the Adobe Flash Player 9 or higher.
Click Here to Get Flash

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
Post a Question / Comment / Request

Allowed tags: i, b, u, sup, sub

Please log into your account at the top of this page or

Create a New Account.

Sign your name and post:

Email:

Post Comment

Name:

OR

06/21/2008 3:48:55 PM
shivali responded with a statement of type: Neutral

why did the negative control also showed amplification...

Reply to This
0
0
            
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.

Reply to This
0
0
                        
08/31/2010 4:10:12 PM
SIAL SAW responded with a statement of type: Agree
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.
Reply to This
0
0
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.

Reply to This
0
0
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

Reply to This
0
0
            
08/31/2010 4:35:00 PM
SIAL SAW responded with a statement of type: Neutral
We have not developed a protocol for Gram-Positive Bacteria currently.
Reply to This
0
0
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?

Reply to This
0
0
            
08/31/2010 4:17:43 PM
SIAL SAW responded with a statement of type: Neutral
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.
Reply to This
0
0
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.
Reply to This
0
0
            
08/31/2010 4:25:20 PM
SIAL SAW responded with a statement of type: Neutral
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.
Reply to This
0
0
02/15/2010 3:17:23 PM
Mike responded with a statement of type: Question
Does anyone know post-digestion of product, what the precipitate is at the bottom?
Reply to This
0
0
            
08/31/2010 4:31:08 PM
SIAL SAW responded with a statement of type: Neutral
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.
Reply to This
0
0
08/05/2010 5:06:20 PM
Anonymous responded with a statement of type: Neutral
Is this discussion still alive?
Reply to This
0
0
            
08/31/2010 4:31:43 PM
SIAL SAW responded with a statement of type: Neutral
Yes
Reply to This
0
0

Mailer.URL:

http://www.jove.com/index/details.stp?id=636

 

Free Access

to this article is sponsored by:

Sponsorship Disclaimer
Publication Criteria

Info

01/22/2008

Publish Date

30528 

Views

 14 

Comments

doi: 10.3791/636 

Tracking

You must be logged in to track comments.

Share this article:

 Email this Article

My Stuff (Please log in to use)

Store a note

Make a Suggestion

Is there a technique you would like to see published?  Click the button above to make a suggestion.

 

 

About Jove

 

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

 

ISSN 1940-087X

 

 

Quick links

 

* Welcome  

* Browse  

* Submit  

* About  

* Editorial Board  

* Press  

* Advertising

* Contact  

* Policies

* Article Index

* Sponsors

* Press Access

* Troubleshooting

* Minerva

 

 

* Twitter.com/JoveJournal

* FaceBook

Subscribe

You don't have to create an account to receive email notifications. 

Please enter your email:

Submit

Copyright (C) JoVE 2006-2008 All Rights Reserved.  Powered by TARGET.  JoVE Site Version 1.893