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.
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.
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.
|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.
|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|