Extract-N-Amp™ Plant PCR Kits Protocol

Catalog Numbers: XNAP2, XNAP2E, and XNAR

• Products
• Reagents and Equipment Required But Not Provided
• Precautions and Disclaimer
• Storage
• Procedure

The Extract-N-Amp™ Plant PCR Kits contain all of the reagents required 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 the 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 the 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 Extract-N-Amp™ PCR Reaction Mix and user provided PCR primers to amplify target DNA.

The Extract-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. This formulation uses JumpStart™ Taq antibody for specific hot start amplification, but does not contain the inert red dye found in the REDExtract-N-Amp™ PCR ReadyMix to allow detection of PCR products by methods that are sensitive to the red dye.

Reagents and Equipment Required But Not Provided

• Paper punch
  
• Forceps (small to medium in size)
• Heat block or water bath at 95 °C
  
• PCR Primers
  
• Water, PCR reagent (W1754)

Precautions and Disclaimer

The Extract-N-Amp™ Plant PCR Kits are for laboratory use only. Not for drug, household or other uses. Consult the MSDS for information regarding hazards and safe handling practices.

Storage

The Extraction Solution, Dilution Solution and Extract-N-Amp™ PCR ReadyMix™ can be stored at 2-8 °C on a short-term basis, but for long-term storage, –20 °C is recommended. Do not store in a “frost-free" freezer.

Procedure

All steps are carried out at room temperature unless otherwise noted.

A. DNA Extraction

1. Rinse the paper punch and forceps in 70% ethanol prior to use and between the handling of different samples.
2. Punch a 0.5 to 0.7 cm disk of leaf tissue into a 2 mL collection tube or suitable vessel using a standard one-hole paper punch. If frozen plant tissue is used, keep the leaves on ice while punching disks.
3. Add 100 µL of the Extraction Solution to the collection tube. Close the tube and vortex briefly. Make sure the disk is covered by the Extraction Solution.
4. Incubate at 95 °C for 10 minutes. Note that leaf tissues usually do not appear to be degraded after this treatment.
5. Add 100 µL of the Dilution Solution and vortex to mix.
6. Store the diluted leaf disk at 2-8 °C. It is not necessary to remove the leaf disk before storage.

B. PCR Amplification

The Extract-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.

1. Add the following reagents to a thin-walled PCR microcentrifuge tube or plate:

*Note: The Extract-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.

2. Mix gently and briefly centrifuge to collect all the components at the bottom of the tube.
3. For thermal cyclers without a heated lid, add 20 µL of mineral oil to the top of each tube to prevent evaporation.
4. 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 Buffer (G2526).

Note: PCR products can be purified, if desired, for downstream applications such as sequencing with the GenElute™ PCR Clean-Up Kit (NA1020).

NOTICE TO PURCHASER: LIMITED LICENSE

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.

GeneElute, Extract-N-Amp, JumpStart , REDExtract-N-Amp and ReadyMix are trademarks of Sigma-Aldrich Co. LLC

JC,RC,PHC 01/13-1