The SYBR® Green Extract-N-Amp™ Tissue PCR Kit contains all the reagents needed for rapid extraction, amplification and detection of genomic DNA from mouse tails and other animal tissues, buccal swabs, hair shafts, and saliva. DNA is rapidly extracted from a tissue by incubating the sample with a mixture of the Extraction Solution and the Tissue Preparation Solution at room temperature for 10 minutes. There is no need for freezing tissue in liquid nitrogen, mechanical disruption, organic extraction, column purification or precipitation of the DNA. After a 3-minute heat denaturing step, an equal volume of Neutralization Solution B is added to the extract to neutralize inhibitory substances and the extract is ready for real-time PCR in any plate-based real-time thermal cycling system.
An aliquot of the neutralized extract is then combined with the Extract-N-Amp™ SYBR® Green PCR ReadyMix™ and user-provided PCR primers. The Extract-N-Amp™ SYBR® Green PCR ReadyMix is a 2X reaction mix containing SYBR® Green, buffer, salts, dNTPs, Taq polymerase and JumpStart™ Taq antibody. It is optimized specifically for use with the extraction reagents and contains JumpStart Taq antibody for hot start PCR to enhance specificity and SYBR® Green I to act as a nonspecific reporter for realtime PCR.
The Extraction, Tissue Preparation & Neutralization Solutions can be stored at 2-8 °C up to 3 weeks; if storing longer than 3 weeks, keep the solutions at -20 °C. Do not store in a "frost-free" freezer.
The Extract-N-Amp™ SYBR® Green PCR ReadyMix should always be kept at –20 °C and out of light as much as possible. Excessive freeze-thawing of the ReadyMix can be detrimental to product performance. Aliquot the ReadyMix into suitably sized portions if necessary to avoid more than five freeze-thaws.
Primer Design & Optimization
SYBR® Green I will detect both specific and non-specific PCR amplicons. Well-designed primers are recommended to ensure the highest possible specificity. Primers for PCR should be designed with the aid of primer design software to eliminate the complications introduced with primer-dimers, nonspecific hybridization and secondary structures. The size of the PCR target should preferably be less than 500 bp, although this product has performed with targets up to 1000 bp. Larger targets are often harder to quantify via real-time PCR.
The primer concentration and cycling parameters need to be optimized and will depend on the system being used. Typical final primer concentrations are ~0.4 µM each. Lower primer concentrations may decrease the accumulation of primer-dimer formation and nonspecific product formation.
For further information on primer design and optimization, consult the manufacturer of your thermalcycling system for assistance.
A positive control is always helpful to verify that the PCR reaction is performing properly. Use purified genomic DNA from the same species as the extract and dilute to 1-5 ng/μL with a 50:50 mixture of Extraction and Neutralization Solutions or Extract-N-Amp™ PCR Diluent (Catalog Number E8155). Do not use water to dilute the positive control. Replace the 4 μL of extract with 4 μL of the positive control in a 20 μL PCR reaction. See Section C., Step 1.
A negative control is necessary to determine if contamination or primer-dimer formation is present. Replace the 4 μL of extract with 4 μL of a 50:50 mixture of Extraction and Neutralization Solutions or Extract-N-Amp™ PCR Diluent in a 20 μL PCR reaction. See Section C., Step 1. Do not use water as a negative control.
All steps are carried out at room temperature unless otherwise noted. The Extraction, Neutralization and Tissue Preparation solutions should be completely thawed, brought to room temperature and gently mixed before use. The Extract-N-Amp™ SYBR® Green PCR ReadyMix should be thawed, mixed well, but kept on ice until needed.
A. DNA extraction from Mouse Tails, Animal Tissues, Hair, or Saliva
B. DNA extraction for Buccal Swabs
C. Real-Time PCR Amplification
The Extract-N-Amp™ SYBR® Green PCR ReadyMix contains JumpStart Taq antibody for specific hot start amplification. Therefore, PCR reactions can be assembled at room temperature without premature Taq DNA polymerase activity. See reference 1 for general information regarding real-time PCR.
With some tissues or other starting materials, PCR may be inhibited by secondary metabolites in the extract such that little or no detectable PCR product is obtained. Diluting the extract five-, ten- or twenty-fold with a 50:50 mixture of Extraction and Neutralization Solutions or with Extract-N-Amp™ PCR Diluent has been shown to alleviate PCR inhibition in many cases.
Note: The reagents included in this kit were formulated together and are dependent on one another for optimal performance of this product. Therefore, only use the recommended diluents instead of water to dilute extracts.
* If required for real-time PCR instrument.
Alternatively, the total volume of a PCR reaction can be adjusted by scaling the reagents relative to the desired PCR total volume.
When preparing multiple PCR reactions, it may be beneficial to create a master-mix. Generate a volume in excess of the amount required to account for measuring losses. After determining the appropriate volume for all reagents, combine and gently vortex to create master-mix. Aliquot 16 µL of master mix and 4 µL of extract per reaction.
2. Mix & Spin Down
Mix gently and briefly centrifuge to collect all components at the bottom of the tube or plate.
3. Amplification Parameters
The amplification parameters should be optimized for individual primers, template, and thermal cyclers
with real-time SYBR® Green detection.
4. Data Analysis
Follow the recommendations of the real-time instrument used to effectively analyze results.
Run a melt curve to ensure the correct PCR target was amplified1. For further verification, run the PCR
reaction on a 2% Agarose-TBE buffered gel.
Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US: 5,994,056 and 6,171,785. The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser’s own internal research. No right under any other patent claim (such as apparatus or system claims in US Patent No. 6,814,934) and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser's activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. Diagnostic uses under Roche patents require a separate license from Roche. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.
SYBR® Green I and its use protected under U.S. Patent No. 5,338,671 and 5,587,287 and corresponding patents in other countries.
SYBR is a registered trademark of Life Technologies
Extract-N-Amp, JumpStart and ReadyMix are trademarks of Sigma-Aldrich Co. LLC
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