REDExtract-N-Amp™ Tissue PCR Kit Protocol

The REDExtract-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 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 reaction mixture 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 and the REDTaq^® dye to allow direct loading of the PCR product onto an agarose gel.

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• Reagents and Equipment Required
• Procedure

Reagents and Equipment Required

Reagents Provided

Reagents	Catalog Number	XNATS 10 Preps, 10 PCRs	XNAT 100 Preps, 100 PCRs	XNATR 1000 Preps, 1000 PCRs
Extraction Solution	E7526	2.5 mL	24 mL	240 mL
Tissue Preparation Solution	T3073	0.3 mL	3 mL	30 mL
Neutralization Solution B	N3910	2.5 mL	24 mL	240 mL
REDExtract-N-Amp™ PCR Reaction Mix This is a 2X PCR reaction mix containing buffer, salts, dNTPs, Taq polymerase, REDTaq® dye, and JumpStart Taq antibody.	R4775	0.15 mL	1.2 mL	12 mL

Equipment Required

• Microcentrifuge tubes (1.5 or 2 mL) or multiwell plate for extractions (200 μL minimal well volume)
• Scissors, micro-dissecting, Catalog No. Z265985
• Forceps (small to medium in size)
• Buccal swab (Sterile foam tipped applicator, Catalog No. A9601
• Sample collection card - Bloodstain card
• Tubes or plate for PCR
• Heat block or thermal cycler at 95 °C
• PCR Primers
• Thermal cycler
• Water, PCR Reagent, Catalog No. W1754
  

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.
    
2. 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 the solution.
   
   Note: For fresh mouse tails, perform extractions within 30 minutes of snipping the tail.
   
   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.
   
   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 the solution. Only one hair shaft, with root, is required per extraction.
   
   For Saliva: Pipette 10 μL of saliva into the solution. Mix thoroughly by vortexing or pipetting.
   
   For Saliva Dried on a Card: Pipette 50 μL of saliva onto a collection card and allow the card to dry. Rinse the hole 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 the solution for incubation period.
    
   
3. Incubate sample at room temperature for 10 minutes. 
   
4. 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.
    
   
5. Add 100μL of Neutralization Solution B to sample and mix by vortexing. 
   
6. 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.
   

Note: Section C, step 1 - 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 can not be recovered efficiently. 
   
2. Pipette 200 μL of Extraction Solution into a 1.5 mL 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 the 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. Continue with Section C, step 1.
   Note: Extracts may be stored at 4 °C for at least 6 months without notable loss in most cases.
   

C. PCR amplification

The REDExtract-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 Reagent	x μL
REDExtract-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 REDExtract-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.                        Perform thermal cycling. The amplification parameters should be
     4. optimized for individual primers, template, and thermal cycler.

	Common Cycling Parameters
Step	Temparature	Time	Cycles
Initial Denaturation	94 °C	3 minutes	1
Denaturation	94 °C	0.5-1 minute	30-35
Annealing	45 to 68 °C	0.5-1 minute
Extension	72 °C	1-2 minutes (~ 1 kb/min)
Final Extension	72 °C	10 minutes	1
Final 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^TM PCR Clean-Up Kit, Catalog Number NA1020.

Troubleshooting

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 REDExtract-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 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 cannot be recovered efficiently.

References

1. Dieffenbach C, Dveksler G. 1995. PCR Primer: A Laboratory Manual. Catalog Number Z364118. 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. Griffin H, Griffin A. 1994. PCR Technology: Current Innovations. Boca Raton: CRC Press.

5. Innis M. 1995. PCR Strategies. Catalog Number Z357499. New York: Academic Press.

6. Innis M. 1990. PCR Protocols: A Guide to Methods and Applications. Catalog Number P8177. San Diego, California: Academic Press.

7. M.J M. 1995. PCR 2: A Practical Approach. Catalog Number Z362387. 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. https://doi.org/10.1101/gr.4.5.s185

10. Erlich HA. 1989. PCR Technology. London: Palgrave Macmillan UK.

NOTICE TO PURCHASER: LIMITED LICENCE

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, 5,618,711, 6,127,155 and claims outside the US corresponding to expired US Patent No. 5,079,352. 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.

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