Extract-N-Amp™ Tissue PCR Kit Protocol

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

Product Description

The Extract-N-Amp™ Tissue PCR Kit contains all the reagents needed to rapidly extract and amplify genomic DNA from mouse tails and other animal tissues, buccal swabs, hair shafts, and saliva. Briefly, the DNA is released from the starting material by incubating the sample with a mixture of the Extraction Solution and the Tissue Preparation Solution at room temperature for 10 minutes. There is no need for mechanical disruption, organic extraction, column purification, or precipitation of the DNA.

After adding Neutralization Solution B, the extract is ready for PCR. An aliquot of the neutralized 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 Reaction Mix is a 2X ready mix containing buffer, salts, dNTPs, and Taq polymerase. It is optimized specifically for use with the extraction reagents. It also contains the JumpStart™ Taq antibody for hot start PCR to enhance specificity, but does not contain the inert red dye found in the REDExtract-N-Amp™ PCR Reaction Mix.

Reagents Provided	Catalog Number	XNAT2 100 Preps, 100 PCRs	XNAT2R 1000 Preps, 1000 PCRs
Extraction Solution	E7526	24 mL	240 mL
Tissue Preparation Solution	T3073	3 mL	30 mL
Neutralization Solution B	N3910	24 mL	240 mL
Extract-N-Amp™ PCR Reaction Mix, This is a 2X PCR reaction mix containing buffer, salts, dNTPs, Taq polymerase, and JumpStart Taq antibody.	E3004	1.2 mL	12 mL

Reagents and Equipment Required But Not Provided

• Microcentrifuge tubes (1.5 or 2 mL) or multiwell plate for extractions (200 µL minimal well volume)
• Small dissecting scissors
• Forceps (small to medium in size)
• Buccal swab - Sterile foam tipped applicator, Catalog Number A9601
• Sample collection card - Bloodstain card, Catalog Number C2613
• Tubes or plate for PCR
• Heat block or thermal cycler at 95 °C
• PCR Primers
• Thermal cycler
• Water, PCR Reagent, Catalog Number W1754

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

The Extract-N-Amp™ Tissue PCR Kit can be stored at 2 to 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. DNA extraction from Mouse Tails, Animal Tissues, Hair, or Saliva

1. Pipette 100 µL of Extraction Solution into a microcentrifuge tube or well of a multiwell plate. Add 25 µL of Tissue Preparation Solution to the tube or well 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 4:1 up to 2 hours before use.
   1. For Fresh or Frozen Mouse Tails: Rinse the scissors and forceps in 70% ethanol prior to use and between different samples. Place a 0.5-1 cm piece of mouse tail tip (cut end down) into the solution. Mix thoroughly by vortexing or pipetting. Ensure the mouse tail is in solution. Note: For fresh mouse tails, perform extractions within 30 minutes of snipping the tail.
   2. For Animal tissues: Rinse the scissors or scalpel and forceps in 70% ethanol prior to use and between different samples. Place a 2–10 mg piece of tissue into the solution. Mix thoroughly by vortexing or pipetting. Ensure the tissue is in the solution.
   3. For Hair Shafts: Rinse the scissors and forceps in 70% ethanol prior to use and between different samples. Trim excess off of the hair shaft leaving the root and place sample (root end down) into solution. Only one hair shaft, with root, is required per extraction.
   4. For Saliva: Pipette 10 µL of saliva into the solution. Mix thoroughly by vortexing or pipetting.
   5. For Saliva Dried on Card: Pipette 50 µL of saliva onto collection card and allow the card to dry. Rinse the punch in 70% ethanol prior to use and between different samples. Punch a disk (preferably 1/8 inch or 3 mm) out of the card from the area with the dried saliva sample. Place disk into the solution. Tap tube or plate on hard surface to ensure disk is in solution for incubation period.
2. Incubate sample at room temperature for 10 minutes.
   
3. Incubate sample at 95 °C for 3 minutes.
   Note: Tissues will not be completely digested at the end of the incubations. This is normal and will not affect performance.
   
4. Add 100 µL of Neutralization Solution B to sample and mix by vortexing.
   
5. Store the neutralized tissue extract at 4 °C or use immediately in PCR. Continue with Section C, step 1.
   Note: For long-term storage, remove the undigested tissue or transfer the extracts to new tubes or wells. Extracts may now be stored at 4 °C for at least 6 months without notable loss in most cases.

B. DNA extraction for Buccal Swabs

1. Collect buccal cells on swab and allow the swab to dry. Drying time is approximately 10 to 15 minutes.
   Note: Due to the low volume of solution used for DNA extraction, a foam-tipped swab should be used. Swabs with fibrous tips, such as cotton or dacron, should be avoided because the solution cannot be recovered efficiently.
2. Pipette 200 µL of Extraction Solution into a microcentrifuge tube. Add 25 µL of Tissue 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 8:1 up to 2 hours before use.
   
3. Place dried buccal swab into solution and incubate at room temperature for 1 minute.
   
4. Twirl swab in solution 10 times and then remove excess solution from the swab into the tube by twirling swab firmly against the side of the tube. Discard the swab. Close the tube and vortex briefly.
   
5. Incubate sample at room temperature for 10 minutes.
   
6. Incubate sample at 95 °C for 3 minutes.
   
7. Add 200 µL of Neutralization Solution B to sample and mix by vortexing.
   
8. Store the neutralized extract at 4 °C or use immediately in PCR.
   Note: Extracts may be stored at 4 °C for at least 6 months without notable loss in most cases.

C. DNA Extraction from Drosophila Tissue

 The Drosophila procedure is very similar to the typical protocol using the Extract-N-Amp™ Tissue PCR Kit and the protocol depends on the number of flies used per experiment.

1. In a 1.5 µL microcentrifuge tube, For 30 fly extract: Crush 30 anesthetized flies in 200 µL of Extraction Solution. Add 50 µL of Tissue Preparation Solution. Vortex. For 10 fly extract: Crush 10 anesthetized flies in 100 µL of Extraction Solution. Add 25 µL of Tissue Preparation Solution. Vortex. For single fly extract: Crush 1 anesthetized fly in 10 µL of Extraction Solution. Add 2.5 µL of Tissue Preparation Solution. Vortex.
2. Incubate at room temperature for 10 minutes.
3. Incubate at 95 °C for 3 minutes.
4. Add 200 µL (for 30 fly extract) or 100 µL (for 10 fly extract) or 10 µL (for single fly extract) of Neutralization Solution B to sample and vortex.
5. Store the neutralized extract at 4 °C or use immediately in PCR.

D. DNA Extraction from C. elegans Worm

The procedure is very similar to the typical protocol using the Extract-N-Amp™ Tissue PCR Kit and the protocol depends on the number of worms used per experiment.

1. For 1 worm: Pipette 20 µL of Extraction Solution into a microcentrifuge tube or well of a multiwell plate. Add 5 µL of Tissue Preparation Solution to the tube or well and pipette up and down to mix.
   For 10 worms: Pipette 100 µL of Extraction Solution into a microcentrifuge tube. Add 25 µL of Tissue Preparation Solution to the tube and pipette up and down to mix. 
   Note:  If several extractions are to be performed, sufficient volumes of Extraction and Tissue Preparation Solutions may be pre-mixed in a ratio of 4:1 up to 2 hours before use.
2. Use your method of choice for picking up a single worm. Place the worm/worms into the solution made in step 1. Mix thoroughly by vortexing. Worm DOES NOT need to be crushed or mashed in tube.
3. Incubate sample at room temperature for 10 minutes.
4. Incubate sample at 95 °C for 3 minutes.
   Note: Worm/worms may not be completely digested at the end of the incubations. This is normal and will not affect performance.
5. Add 20 µL (for 1 worm) and 100 µL (for 10 worms) of Neutralization Solution B to sample and mix by vortexin
6. Store the neutralized tissue extracts at 4 °C or use immediately in PCR.

PCR amplification

The Extract-N-Amp™ PCR Reaction Mix 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 approximately 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 grade	x µL
Extract-N-Amp™ PCR reaction mix	10 µL
Forward primer	y µL
Reverse primer	y µL
Tissue extract	4 µL*
Total volume	20 µL

*Note: The Extract-N-Amp™ PCR Reaction Mix is formulated to compensate for components in the Extraction, Tissue Preparation, and Neutralization Solutions. If less than 4 μL of tissue extract is added to the PCR reaction volume, use a 50:50 mixture of Extraction:Neutralization B Solutions to bring the volume of tissue extract up to 4 µL.

2. Mix gently.

3. For thermal cyclers without a heated lid, add 20 µL of mineral oil on top of the mixture in each tube to prevent evaporation.

4. Perform thermal cycling. 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 Solution, Catalog Number G2526.

The same protocol may be followed using REDExtract-N-Amp™ Tissue PCR Kit (XNAT) or SYBR^® Green Extract-N-Amp™ Tissue PCR Kit (XNATG, XNATRG). If using REDExtract-N-Amp™ Tissue PCR Kit, the amplified DNA can be loaded directly onto an agarose gel after the PCR is completed without the need to add a separate loading buffer/tracking dye.

For real-time PCR using SYBR^® Green Extract-N-Amp™ Tissue PCR Kit (XNATG, XNATRG), amplification parameters should be optimized for individual primers, template, and thermal cycler.
Reagents to be added to a thin-walled PCR microcentrifuge tube or plate:

Reagent	Volume
Water, PCR grade	x µl
SYBR® Green Extract-N-Amp™ PCR reaction mix	10 µl
Forward primer	y µl
Reverse primer	y µl
Reference dye	z µl*
Extract	4 µl**
Total volume	20 µl

*If required for real-time PCR instrument.
**The SYBR^® Green Extract-N-Amp™ PCR Reaction Mix is formulated to compensate for components in the Extraction, Tissue Preparation, and Neutralization Solutions. If less than 4 μL of tissue extract is added to the PCR reaction volume, use a 50:50 mixture of Extraction: Neutralization B Solutions to bring the volume of tissue extract up to 4 µL.

Common cycling parameters for real-time PCR:

Step	Temperature	Time	Cycles
Initial Denaturation	95 °C	3 minutes	1
Denaturation	95 °C	15 – 30 seconds
Annealing	45 to 68 °C	15 seconds -1 minute	35 - 45
Melt Curve (as applicable)	As applicable °C	As applicable

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

Troubleshooting Guide

Problem	Cause	Solution
Little or no PCR product is detected.	PCR reaction may be inhibited due to contaminants in the tissue extract.	Dilute the tissue extract with a 50:50 mix of Extraction and Neutralization 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 tissue extract.
	Extraction is insufficient.	Incubate samples at 55 °C for 10 minutes instead of room temperature.
	A PCR component may be missing or degraded.	Run a positive control to ensure 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 in 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 in 1 °C increments.
	The denaturation time may be too long or too short.	Optimize the denaturation time by increasing or decreasing it in 10 second increments.
	The extension time may be too short.	Increase the extension time in 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, Catalog Number B0300, 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 Reaction Mix. 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” result.	Reagents are contaminated.	We recommend 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.
Tissue is not digested after incubations.	Tissue is not expected to be completely digested.	The REDExtract-N-Amp™ Tissue PCR Kit does not require the tissue to be completely digested. Sufficient DNA is released for PCR without completely digesting the tissue.
Buccal swab absorbed all the solution.	The recommended type of swab was not used.	Due to the low volume of solution used for DNA extraction, a foam tipped swab should be used. Swabs with fibrous tips, such as cotton or dacron, should be avoided because the solution can not be recovered efficiently.

References

1. Dieffenbach C, Dveksler G(. 1995. PCR Primer: A Laboratory Manual. 2nd. 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. 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. Roux KH. 1995. Optimization and troubleshooting in PCR.. Genome Research. 4(5):S185-S194. https://doi.org/10.1101/gr.4.5.s185

5. Griffin H, Griffin A(. 1994. PCR Technology: Current Innovations. Boca Raton, FL: CRC Press.

6. Innis M. 1995. PCR Strategies. New York: Academic Press.

7. Innis M. 1990. PCR Protocols: A Guide to Methods and Applications. San Diego, California: Academic Press.

8. Saiki RK. 1989. The Design and Optimization of the PCR.7-16. https://doi.org/10.1007/978-1-349-20235-5_1

9. McPherson M, (Eds) ea. 1995. PCR 2: A Practical Approach. New York: IRL Press.

10. Newton C(. 1995. PCR: Essential Data. New York: John Wiley & Sons.

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.

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

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