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View additional product information for Phire Tissue Direct PCR Master Mix - FAQs (F170L, F170S)
14 product FAQs found
In order to obtain best results from Direct PCR, it is important to use a very small amount of starting materials. The recommended size is 0.3-0.5 mm in diameter. We recommend using Tissue Puncher 0.3 mm (Cat. No. F-200S), which is a convenient tool for sampling and is suitable for both plant and animal tissues.
It is very important to include a control reaction in the Direct PCR to ensure that the PCR conditions are optimal for the specific tissue or plant material used. Control primers are universal primers that amplify a small fragment from a conserved genome region. If the reaction with control primers fails, the sample material may not be suitable for Direct PCR, or PCR conditions need optimization.
With the Phire Tissue Direct PCR Master Mix you can amplify DNA from differently stored tissues (fresh, frozen, FFPE) from human and a variety of animal species.
You can test any plant or tissue sample. However, it is recommended to first optimize the reaction. When working with new sample materials, start with the Dilution & Storage protocol as it allows several PCR reactions to be performed from the same sample during optimization. It is recommended to perform a positive control with purified sample DNA to ensure that the PCR conditions are working. If the positive control with purified DNA fails, the PCR conditions should be optimized before continuing further.
Yes, the Direct PCR Master Mixes are upgrades of the Direct PCR kits and thus are suitable for the same sample materials.
No, tissue punchers are not provided. You can purchase 0.3 mm Tissue Punchers (Cat. No. F-200S), or cut the sample with a scalpel. Some scientists use 100 µL pipette tips to take a sample.
Yes, similar to the Direct PCR Kits, Direct PCR Master Mixes work in both Direct and Dilution & Storage protocols. Direct PCR from blood is performed with Direct protocol only.
FFPE tissue, FFPE tissue sections, tissue blocks and microscope slides can all be used in sample preparation.
Direct PCR Kits and Master Mixes employ Phire Hot Start II DNA Polymerase or Phusion Hot Start II Polymerase (depending on the kit), that both possess the following activities: 5'-3' DNA polymerase activity and a weak 3'-5' exonuclease activity. When cloning fragments amplified with these DNA Polymerases, blunt end cloning is recommended. If TA cloning is required, it can be performed by adding A overhangs to the blunt PCR product. However, before adding the overhangs, it is very important to remove all the DNA polymerase by purifying the PCR product carefully. Any remaining Phusion or Phire DNA Polymerases will degrade the A overhangs, creating blunt ends again. All Direct PCR kits are compatible with sequencing techniques. The green dye present in the Direct Master Mix kits does not interfere with the sequencing reactions.
When amplifying DNA directly from mouse tissue, it is important to add DNARelease Additive before gel electrophoresis as otherwise cell debris present in the PCR products can cause DNA fragments to get trapped in the agarose gel wells. Add 1.5 µL of DNARelease Additive into a 50 µL PCR reaction. If you are using Phire Animal Tissue Direct PCR Kit add the DNARelease Additive to the loading dye for gel electrophoresis. Mix 100 µL of DNARelease Additive with 1 mL of gel loading dye. The dilution is stable for 2 weeks at +4 degrees C. For long term use, store the dilution at -20 degrees C. Before agarose gel electrophoresis, add 15 µL of the pre-mixed gel loading dye into a 50 µL PCR reaction.
After PCR, it is recommended to centrifuge the PCR reactions at 1000 x g for 1-3 minutes to collect the supernatant for subsequent analysis. We recommend storing the supernatant at -20 degrees C
The optimal length for Direct PCR from liquid samples is up to 5 kb and from solid samples up to 1 kb. However, the maximum length depends on the sample type, sample size and the reaction volume. For example we have amplified a 7.5 kb fragment directly from human blood and tissue samples, and a 3 kb fragment from mouse ear and 5 kb fragment from plant leaves.
DNARelease Additive is required when PCR is performed directly from certain tissue samples using the Direct protocol. Cell debris present in these PCR products can cause DNA fragments to get trapped in the agarose gel wells. DNARelease Additive eliminates this problem and should be added directly into the PCR reaction. Please reduce the amount of water used or simply add the DNARelease to the PCR reaction setup. DNARelease is also used in the Dilution & Storage protocol to improve the release of DNA from the tissue sample.
Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Applications Support Center.
Yes, we recommend using Phire Tissue Direct PCR Master Mix (Cat. No. F170S, F170L).