The Extract-N-Amp™ Tissue PCR Kit contains all the reagents needed to rapidly extract and amplify genomic DNA from mouse tails and other animal tissues, buccal swabs, hair shafts, and saliva. Briefly, the DNA is released from the starting material 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 mechanical disruption, organic extraction, column purification, or precipitation of the DNA.
After adding Neutralization Solution B, the extract is ready for PCR. An aliquot of the neutralized extract is then combined with the Extract-N-Amp™ PCR Reaction Mix and user-provided PCR primers to amplify target DNA. The Extract-N-Amp™ PCR Reaction Mix is a 2X ready mix containing buffer, salts, dNTPs, and Taq polymerase. It is optimized specifically for use with the extraction reagents. It also contains the JumpStart™ Taq antibody for hot start PCR to enhance specificity, but does not contain the inert red dye found in the REDExtract-N-Amp™ PCR Reaction Mix.
All steps are carried out at room temperature unless otherwise noted.
The Drosophila procedure is very similar to the typical protocol using the Extract-N-Amp™ Tissue PCR Kit and the protocol depends on the number of flies used per experiment.
The procedure is very similar to the typical protocol using the Extract-N-Amp™ Tissue PCR Kit and the protocol depends on the number of worms used per experiment.
The Extract-N-Amp™ PCR Reaction Mix contains JumpStart Taq antibody for specific hot start amplification. Therefore, PCR reactions can be assembled at room temperature without premature Taq DNA polymerase activity.
Typical final primer concentrations are approximately 0.4 µM each. The optimal primer concentration and cycling parameters will depend on the system being used.
1. Add the following reagents to a thin-walled PCR microcentrifuge tube or plate:
*Note: The Extract-N-Amp™ PCR Reaction Mix is formulated to compensate for components in the Extraction, Tissue Preparation, and Neutralization Solutions. If less than 4 μL of tissue extract is added to the PCR reaction volume, use a 50:50 mixture of Extraction:Neutralization B Solutions to bring the volume of tissue extract up to 4 µL.
2. Mix gently.
3. For thermal cyclers without a heated lid, add 20 µL of mineral oil on top of the mixture in each tube to prevent evaporation.
4. Perform thermal cycling. The amplification parameters should be optimized for individual primers, template, and thermal cycler.
Common cycling parameters:
5. The amplified DNA can be loaded onto an agarose gel after the PCR is completed with the addition of a separate loading buffer/tracking dye such as Gel Loading Solution, Catalog Number G2526.
The same protocol may be followed using REDExtract-N-Amp™ Tissue PCR Kit (XNAT) or SYBR® Green Extract-N-Amp™ Tissue PCR Kit (XNATG, XNATRG). If using REDExtract-N-Amp™ Tissue PCR Kit, the amplified DNA can be loaded directly onto an agarose gel after the PCR is completed without the need to add a separate loading buffer/tracking dye.
For real-time PCR using SYBR® Green Extract-N-Amp™ Tissue PCR Kit (XNATG, XNATRG), amplification parameters should be optimized for individual primers, template, and thermal cycler.
Reagents to be added to a thin-walled PCR microcentrifuge tube or plate:
*If required for real-time PCR instrument.
**The SYBR® Green Extract-N-Amp™ PCR Reaction Mix is formulated to compensate for components in the Extraction, Tissue Preparation, and Neutralization Solutions. If less than 4 μL of tissue extract is added to the PCR reaction volume, use a 50:50 mixture of Extraction: Neutralization B Solutions to bring the volume of tissue extract up to 4 µL.
Common cycling parameters for real-time PCR:
Note: PCR products can be purified, if desired, for downstream applications such as sequencing with the GenElute™ PCR Clean-Up Kit, Catalog Number NA1020
Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US: 5,789,224 and 5,618,711. 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, no right to perform any patented method, 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.
JumpStart and JumpStart Antibody are licensed under U.S. Patent No. 5,338,671 and 5,587,287 and corresponding patents in other countries.
Extract-N-Amp, GenElute, JumpStart and REDExtract-N-Amp are trademarks of Sigma-Aldrich Co. LLC
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