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Molecular Biology

Extract-N-Amp™ FFPE Tissue PCR Protocol

REDExtract-N-Amp™ Tissue PCR Kit Protocol, for formalin-fixed paraffin embedded sections

The following procedure is a modification of the Extract-N-Amp™ Tissue PCR protocol. Other DNA starting samples (49 kb PDF) may also be used. Download the full REDExtract-N-Amp™ Tissue PCR Kit Protocol (49 kb PDF) of the original protocol for a complete list.



Click the image to see a researcher use the Sigma Extract-N-Amp kit for mouse tails and scroll below to see a modification of the same protocol for FFPE samples.


 

Product Numbers XNATS, XNAT, and XNATR


Note:
The quality of DNA present in formalin-fixed, paraffin-embedded tissues can be variable depending on a number of factors among which include the age of the sample, the treatment of the sample before fixation, the fixation time and fixation conditions. Fixed samples in some cases may not contain DNA of sufficient quality for many downstream applications including amplification with the Extract N Amp Tissue PCR kit.


DNA extraction from FFPE Animal tissues:
All steps are carried out at room temperature unless otherwise noted.


  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. Carefully slice a small section of paraffin embedded tissue out of your sample, then scrape/slice excess paraffin from sample. Weigh approximately 5 mg of the tissue sample, and place in a 1.5 ml microcentrifuge tube (not provided).
  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 100 µl 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.

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 Grade 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.
  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 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
    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.




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