REDExtract-N-Amp™ Seed PCR Protocol

Catalog Numbers: XNASS, XNAS, and XNASR

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 Provided Product No.
XNASS
10 extractions,
10 amplifications
XNAS
100 extractions,
100 amplifications
XNASR
1000 extractions,
1000 amplifications
Extraction Solution E7526 1.2 ml 6 ml 60 ml
Seed Preparation Solution S1193 0.15 ml 0.9 ml 6 ml
Neutralization Solution B N3910 1.2 ml 6 ml 60 ml
REDExtract-N-Amp PCR ReadyMix
This is a 2X PCR mix containing buffer, salts, dNTPs, Taq polymerase, REDTaq dye and JumpStart Taq antibody
R4775 0.15 ml 1.2 ml 12 ml

Reagents and Equipment Required But Not Provided

Items common to all procedures:

  • Tubes or plates for PCR
  • PCR primers
  • Thermal cycler
  • Water, PCR grade, Catalog Number W1754        

For individual 1.5 ml tubes:

  • 1.5 ml microcentrifuge tubes
  • Heat block or thermal cycler
  • Disposable plastic pestles, Catalog Number Z359947

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
  • Optional: Heat block with 96 well block

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 long term 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 mL for soybean or similar sized seeds
  • 600 mL for cotton or similar sized seeds
  • 200 mL for canola, sorghum, wheat, or similar sized seeds
  • 100 mL 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 mL for soybean or similar sized seeds
  • 600 mL for corn or similar sized seeds
  • 400 mL for cotton or similar sized seeds
  • 100 mL 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:

 

Reagent Volume
Water, PCR Reagent X µL
REDExtract-N-Amp PCR ReadyMix 10 µL
Forward Primer Y µL
Reverse Primer Y µL
Seed extract 4 µL*
Total Volume 20 µL

*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:

 

Step Temperature Time Cycles
Initial Denaturation 94 °C 3 min 1
Denaturation 94 °C 0.5-1 min 30-35
Annealing 45 to 68 °C 0.5-1 min
Extension 72 °C 1-2 min (~1kb/min)
Final Extension 72 °C 10 min 1
Hold 4 °C indefinitely  

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

 

Problem Cause Solution
Little or no PCR product is detected Seeds were not ground sufficiently For 96 well grind: Increase grinding time in bead mill.  For seeds with a tough seed coat, it is helpful to break the seed before putting it into the plate to grind. For individual 1.5 ml tube: For seeds with a tough seed coat, it is helpful to break the seed before incubating at 55°C.
PCR reaction may be inhibited due to contaminants in the plant extract. Dilute the extract with a 50:50 mix of extraction and dilution solutions. To test for inhibition, include a DNA control and/or spike a known amount of template (100-500 copies) into the PCR along with the plant extract.
A PCR component may be missing or degraded. Run a positive control to insure that components are functioning. A checklist is also recommended when assembling reactions.
There may be too few cycles performed. Increase the number of cycles (5-10 additional cycles at a time).
The annealing temperature may be too high. Decrease the annealing temperature by 2-4 °C increments.
The primers may not be designed optimally. Confirm the accuracy of the sequence information. If the primers are less than 22 nucleotides long, try to lengthen the primer to 25-30 nucleotides. If the primer has a GC content of less than 45%, try to redesign the primer with a GC content of 45-60%.
The denaturation temperature may be too high or too low. Optimize the denaturation temperature by increasing or decreasing the temperature by 1 °C increments.
The denaturation time may be too long or too short. Optimize the denaturation time by increasing or decreasing it by 10 second increments.
The extension time may be too short. Increase the extension time by 1 minute increments, especially for long templates.
Target template is difficult. In most cases, inherently difficult targets are due to unusually high GC content and/or secondary structure. Betaine (Product Code B 0300) has been reported to help amplification of high GC content templates at a concentration of 1.0-1.7 M.
Multiple products JumpStart Taq antibody is not working correctly. Do not use DMSO or formamide with Extract-N-Amp PCR ReadyMix. It can interfere with the enzyme-antibody complex. Other cosolvents, solutes (e.g., salts) and extremes in pH or other reaction conditions may reduce the affinity of the JumpStart Taq antibody for Taq polymerase and thereby compromise its effectiveness.
Touchdown PCR may be needed. “Touchdown” PCR significantly improves the specificity of many PCR reactions in various applications. Touchdown PCR involves using an annealing/extension temperature that is higher than the Tm of the primers during the initial PCR cycles. The annealing/extension temperature is then reduced to the primer Tm for the remaining PCR cycles. The change can be performed in a single step or in increments over several cycles.
Negative Control shows a PCR product or “false positive” results are obtained Reagents are contaminated. Sigma recommends that a reagent blank without DNA template be included as a control in every PCR run to determine if the reagents used in extraction or PCR are contaminated with a template from a previous reaction.

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.

Materials

     

References

  1. Dieffenbach, C.W. and Dveksler, G.S. (Eds.) PCR Primer: A Laboratory Manual, 2nd ed., (Cold Spring Harbor Laboratory Press, New York, 2003). Catalog Number Z701270
  2. Don, R.H., et al., ‘Touchdown' PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res., 19, 4008 (1991).
  3. Erlich, H.A. (Ed.) PCR Technology: Principles and Applications for DNA Amplification (Stockton Press, New York, 1989).
  4. Griffin, H.G. and Griffin, A.M. (Eds.) PCR Technology: Current Innovations (CRC Press, Boca Raton, FL, 1994).
  5. Innis, M.A., et al. (Eds.) PCR Strategies, Academic Press, New York, 1995).
  6. Innis, M., et al. (Eds.) PCR Protocols: A Guide to Methods and Applications (Academic Press, San Diego, CA, 1990).
  7. McPherson, M.J. et al. (Eds.) PCR 2: A Practical Approach (IRL Press, New York, 1995).
  8. Newton, C.R.(Ed.) PCR: Essential Data (John Wiley & Sons, NY, 1995)
  9. Roux, K.H. Optimization and troubleshooting in PCR. PCR Methods Appl., 4, 5185-5194 (1995).
  10. Saiki, R., PCR Technology: Principles and Applications for DNA Amplification (Stockton, NY, 1989).

 

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.

GenElute, JumpStart and REDExtract-N-Amp are trademarks, and REDTaq is a registered trademark, of Sigma-Aldrich Co. LLC.

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