REDExtract-N-Amp™ Tissue PCR Kit Protocol

Product Nos. XNATS, XNAT, XNATR

Overview

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.

Reagents and Equipment Required, Not Provided:

• 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, Catalog No. C2613
• 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:

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.

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.

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.