Product Nos. XNATS, XNAT, XNATR
The REDExtract-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 REDExtract-N-Amp™ PCR Reaction Mix and user-provided PCR primers to amplify target DNA. The REDExtract-N-Amp™ PCR Reaction Mix is a 2X reaction mixture 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 and the REDTaq® dye to allow direct loading of the PCR product onto an agarose gel.
All steps are carried out at room temperature unless otherwise noted.
Note: Section C, step 1 - For long term storage, remove the undigested tissue or transfer the extracts to new tubes or wells. Extracts may now be stored at 4 °C for at least 6 months without notable loss in most cases.
The REDExtract-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 REDExtract-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. Perform thermal cycling. The amplification parameters should be
4. optimized for individual primers, template, and thermal cycler.
5. The amplified DNA can be loaded directly onto an agarose gel after the PCR is completed. It is not necessary to add a separate loading buffer/tracking dye.
Note: PCR products can be purified, if desired, for downstream applications such as sequencing with the GenEluteTM 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, 5,618,711, 6,127,155 and claims outside the US corresponding to expired US Patent No. 5,079,352. 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.
GenElute, JumpStart and REDExtract-N-Amp are trademarks of Sigma-Aldrich Co. LLC.
REDTaq is a registered trademark of Sigma-Aldrich Co. LLC.
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