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REDExtract-N-Amp Seed PCR Kit Protocol

Catalog Numbers: XNASS, XNAS, and XNASR

Product Description
Reagents and Equipment Required But Not Provided
Precautions and Disclaimer
Storage
Procedure
Troubleshooting Guide
Materials
References

Product Description

The REDExtract-N-Amp Seed PCR Kit contains all the reagents needed to rapidly extract and amplify genomic DNA from seeds (soybean, corn, wheat, etc.). Briefly, DNA is extracted from ground seed material by incubation in a mixture of Extraction Solution and Seed Preparation Solution at 55 °C for 10 minutes. There is no need for organic extraction, column purification, or precipitation of the DNA. After the extraction is stopped by incubation at 95 °C for 3 minutes, an equal volume of Neutralization Solution B is added and 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 ready mix containing buffer, salts, dNTPs, and Taq DNA polymerase. It is optimized specifically for use with the extraction reagents. This formulation also contains the JumpStart™ antibody for specific hot start amplification, and REDTaq® dye to allow direct loading of the PCR product onto an agarose gel.

Reagents and Equipment Required But Not Provided

Items common to all procedures:

For individual 1.5 mL tubes:

For individual 1.5 mL tubes with liquid nitrogen

  • 1.5 mL microcentrifuge tubes
  • Liquid nitrogen
  • Mortar and pestle
  • Heat block or thermal cycler

For 96 well plates:

  • Bead Mill (2000 Geno/Grinder from Spex Certiprep or equivalent)
  • 4 mm stainless steel grinding balls (Spex Certiprep)
  • 2 mL Square well block (Whatman Product Code 7701-5200)
  • 96 well sealing mat (Brinkmann Instruments Product Code 951-03-014-7)
  • 96 well PCR plate
  • Thermal cycler

Precautions and Disclaimer

This product is for R&D use only, not for drug, household, or other uses. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.

Storage

All components of the REDExtract-N-Amp PCR Kit can be stored at 2-8 °C for up to 3 weeks. For longterm storage, greater than 3 weeks, -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. Grinding Seeds
Following are three different methods for grinding seeds.

1. Grind using a Bead Mill
1a. Place 1 seed into each well of a 2 mL square well block.
Notes: With Arabidopsis or similar sized seeds, approximately 50 seeds should be placed in a single well.
This grinding procedure is not recommended for corn seeds, because results with such large, tough
seeds are inconsistent.

1b. Pipette PCR grade water into the well according to the following volumes:

800 µL for soybean or similar sized seeds
600 µL for cotton or similar sized seeds
200 µL for canola, sorghum, wheat, or similar sized seeds
100 µL for Arabidopsis or similar sized seeds

1c. Place a 4 mm stainless steel grinding ball in each well of the 2 mL 96 square well block and cover with
sealing mat. Place block in the bead mill and shake at 1,500 rpm for 10 minutes. Continue to Section B.

2. Grind individually using a plastic pestle

2a. Place 1 seed into a 1.5 mL microcentrifuge tube.

Note: With Arabidopsis or similar sized seeds approximately 50 seeds should be placed in a single tube.

2b. Pipette PCR grade water into the well according to the following volumes:

800 µL for soybean or similar sized seeds
600 µL for corn or similar sized seeds
400 µL for cotton or similar sized seeds
100 µL for Arabidopsis, canola, sorghum, wheat, or similar sized seeds

2c. Incubate the seed with water for 1 hour at 55 °C.

2d. Grind hydrated seeds in tube using a plastic pestle. Continue to Section B.

3. Grind individually using liquid nitrogen

3a. Grind seed into a fine powder in liquid nitrogen using a mortar and pestle.

Note: With small seeds, such as Arabidopsis and canola, more than one seed must be ground to collect
enough ground seed material.

3b. Transfer between 5 and 100 mg of ground seed material into a pre-weighed 1.5 mL microcentrifuge
tube. Record mass of transferred seed material.

3c. Pipette 4 µL of water for every mg of transferred ground seed material into the sample tube and vortex
to mix. Continue to Section B.

B. Extraction of Seeds

1. Pipette 45 µL of Extraction Solution into a 1.5 mL microcentrifuge tube or multiwell PCR plate. Add 5 µL of
Seed Preparation Solution to the tube and pipette up and down to mix.
Note: If several extractions will be performed, sufficient volumes of Extraction and Tissue Preparation Solutions
may be pre-mixed in a ratio of 9:1 up to 2 hours before use. The mixture should then be dispensed in 50 µL
volumes into tubes or multiwell plates.

2. Pipette 5 µL of the ground seed suspension from Section A into the Extraction Solution and Seed Preparation
Solution mixture and vortex or pipette up and down to mix.

3. Incubate the mixture at 55 °C for 10 minutes to extract DNA. Note that the ground seed will not appear to be
digested at the end of this incubation; however, sufficient DNA will be released for PCR.

4. Incubate the mixture at 95 °C for 3 minutes to stop the extraction.
Note: Steps 3 and 4 can be performed in a thermalcycler if using a 96 well PCR plate.

5. Add 50 µL of Neutralization Solution B to the mixture and vortex or pipette up and down to mix.

6. Store the neutralized seed extract at 2-8 °C or continue to Section C.

C. PCR amplification

The REDExtract-N-Amp PCR Reaction Mix contains JumpStart 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 REDExtract-N-Amp PCR Reaction Mix is formulated to compensate for components in the Extraction, Seed Preparation, and Neutralization B Solutions. If less than 4 µL of seed extract is added to the PCR reaction volume, use a 50:50 mixture of Extraction and Neutralization B Solutions to bring the volume of seed extract up to 4 µL.

2. Mix gently and briefly centrifuge to collect all the components to the bottom of the tube.

3. For thermalcyclers 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 Number NA1020

Materials
Loading

References

1.
Stephen AB. 2004 . A-Z of Quantitative PCR International University Line.
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 HA. 1989. PCR Technology. http://dx.doi.org/10.1007/978-1-349-20235-5
4.
Griffin H, Griffin A. 1994 . PCR Technology: Current Innovations Boca Raton. FL CRC Press.
5.
Innis Mea. 1995. PCR Strategies New York Academic Press.
6.
Innis Mea. 1990 . PCR Protocols: A Guide to Methods and Applications . San Diego: CA Academic Press.
7.
McPherson Mea. 1995 . PCR 2: A Practical Approach New York IRL Press.
8.
Newton C. 1995. PCR: Essential Data new york John Wiley & Sons.
9.
Roux KH. 1995. Optimization and troubleshooting in PCR.. Genome Research. 4(5):S185-S194. http://dx.doi.org/10.1101/gr.4.5.s185
10.
Saiki RK. 1989. The Design and Optimization of the PCR.7-16. http://dx.doi.org/10.1007/978-1-349-20235-5_1

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.

JumpStart and JumpStart Antibody are licensed under U.S. Patent No. 5,338,671 and 5,587,287 and corresponding patents in other countries.