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 Provided	Catalog Number	XNAP2 100 extractions, 100 amplifications	XNAP2E 100 extractions, 500 amplifications	XNAR 1,000 extractions, 1,000 amplifications
Extraction Solution	E7526	12 mL	12 mL	120 mL
Dilution Solution	D5688	12 mL	12 mL	120 mL
Extract-N-Amp™ PCR ReadyMix™	E3004	1.2 mL	5 x 1.2 mL	12 mL
Collection Tubes, 2 mL	T5449 or T7813	2 X 50 each	2 X 50 each	Not included

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:

Reagent	Volume
Water, PCR reagent	x µL
Extract-N-Amp™ PCR ReadyMix™	10 µL:
Forward primer	y µL
Reverse primer	y µL
Leaf disk extract	4 µL*
Total volume	20 µL

*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

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

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).

Related Products	Catalog Numbers
Tubes for PCR	Z374873, Z374962 and Z374881
PCR Marker	P9577
Precast Agarose Gels	P6097
TBE Buffer	T4415, T6400 and T9525
Ethanol	E7148, E7023 and 459836

Troubleshooting Guide

Problem	Cause	Solution
Little or no PCR product is detected	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.
Contamination	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.

References

1. Biermann C. 2004. PCR Primer: A Laboratory Manual. Second Edition. Edited by Carl W  Dieffenbach and , Gabriela S  Dveksler. Cold Spring Harbor (New York): Cold Spring Harbor Laboratory Press. $195.00 (hardcover); $140.00 (paper). xi + 520 p; ill.; index. ISBN: 0?87969?653?2 (hc); 0?87969?654?0 (pb). 2003.. The Quarterly Review of Biology. 79(2):204-205. https://doi.org/10.1086/423063

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. https://doi.org/10.1093/nar/19.14.4008

3. Erlich HA. 1989. PCR Technology. https://doi.org/10.1007/978-1-349-20235-5

4. Wilson S. 1995. PCR Technology. Current Innovations: Edited by H. G. Griffin and Annette M. Griffin. 1994. ISBN 0-8493-8674-8. CRC Press Inc., Boca Raton. Pp. 370.  41.00.. Journal of Medical Microbiology. 42(1):74-74. https://doi.org/10.1099/00222615-42-1-74a

5. Innis M. 1995. PCR Strategies. Academic Press, New York.:

6. 1991. PCR Protocols: A guide to methods and applications, edited by Michael A. Innis et al., Academic Press, 1990, 482 pp, $39.95. Mol. Reprod. Dev.. 28(4):419-419. https://doi.org/10.1002/mrd.1080280418

7. McPherson M. 1995. PCR 2: A Practical Approach.. IRL Press, New York.:

8. Newton C. 1995. PCR: Essential Data.. John Wiley & Sons, New York.:

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