Catalog Numbers: XNAPS, XNAP, and XNAPR

Product Description

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 user-provided PCR primers to amplify target DNA. The REDExtract-N-A mp 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.

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, Catalog Number W1754

Precautions and Disclaimer

The REDExtract-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 REDExtract-N-Amp PCR ReadyMix can be stored at 2-8 °C for short-term;
–20 °C for long-term. 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 handling different samples.

2. Punch a 0.5 to 0.7 cm disk of leaf tissue into a 2 mL collection tube 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 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 Dilution Solution and vortex to mix.

6. Store the diluted leaf extract at 2-8 °C. It is not necessary to remove the leaf disk before storage.

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.

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

*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.

2. Mix gently and briefly centrifuge to collect all 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 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 GenElute™ PCR Clean-Up Kit, Catalog No. NA1020.

Troubleshooting Guide

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, 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.

Materials
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References

1.
Diffenbach C, Dveksler G. 2003. PCRPrimer: A Laboratory Manual, 2nd edition.. New York.: Cold Spring Harbor Laboratory Press.
2.
Don R, Cox PT, Wainwright B, Baker K, Mattick JS. 1991. ?Touchdown? PCR to circumvent spurious priming during gene amplification. Nucl Acids Res. 19(14):4008-4008. http://dx.doi.org/10.1093/nar/19.14.4008
3.
Erlich H. 1989. PCR Technology: Principles and Applications for DNA Amplification. Stockton Press, New York.. New York.: Stockton Press,.
4.
Griffin H, Griffin A. 1994. PCR Technology: Current Innovations.. CRC Press, Boca Raton, FL .:
5.
Innis M. 1995. PCR Strategies. New York : Academic Press.
6.
Innis M. 1990. PCR Protocols: A Guide to Methods and Applications. San Diego, California: Academic Press.
7.
McPherson M. 1995. PCR 2: A Practical Approach. New York: IRL Press.
8.
Newton C. 1995. PCR: Essential Data. New York: John Wiley & Sons.