The REDExtract-N-Amp Plant PCR Kits contain all the reagents needed to rapidly extract and amplify genomic DNA from plant leaves. Briefly, the DNA is extracted from a piece of leaf tissue, a 0.5 to 0.7 cm disk cut with a standard paper punch, by incubation in Extraction Solution at 95 °C for 10 minutes. There is no need for freezing plant tissue in liquid nitrogen, mechanical disruption, organic extraction, column purification or precipitation of the DNA. After an equal volume of Dilution Solution is added to the extract to neutralize inhibitory substances, the extract is ready for PCR.
An aliquot of the diluted extract is then combined with the REDExtract-N-Amp PCR ReadyMix™ and userprovided PCR primers to amplify target DNA. The REDExtract-N-Amp PCR ReadyMix is a 2X reaction mix containing buffer, salts, dNTPs, and Taq polymerase. It is optimized specifically for use with the extraction reagents. It also contains JumpStart™ Taq antibody for hot start PCR to enhance specificity and 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.
A. DNA extraction
B. PCR amplification
The REDExtract-N-Amp 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.
Typical final primer concentrations are ~0.4 µM each. The optimal primer concentration and cycling parameters will depend on the system being used.
*Note: The REDExtract-N-Amp PCR ReadyMix is formulated to compensate for components in the Extraction and Dilution Solutions. If less than 4 μL of leaf disk extract is added to the PCR reaction volume, use a 50:50 mixture of Extraction:Dilution Solutions to bring the volume of leaf disk extract up to 4 μL.
Common cycling parameters:
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, nontransferable 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.
GenElute, JumpStart, ReadyMix and REDExtract-N-Amp are trademarks, and REDTaq is a registered trademark, of Sigma-Aldrich Co. LLC.
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