Search
Search
View additional product information for Phire Plant Direct PCR Kit (without sampling tools) - FAQs (F130WH)
11 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.
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.
Yes, frozen (-20 degrees C and -70 degrees C), dried and fresh plant leaves can be used. We have succeeded in amplifying DNA directly from oak leaves stored at -20 degrees C for 10 years. However the highest amplification yields are achieved when using fresh samples.
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.
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.
No, we do not offer a sampling tool for using with the Phire Plant Direct PCR Kit.
Standard biopsy punch products can be used as alternative.
In the Phire Plant Direct PCR Kit User Guide, section 4 lists the following recommendations:
- We recommend using a puncher that is 0.5 mm or 0.35 mm in diameter.
- Other ways to take a sample is by cutting with scalpel to obtain 0.35–0.50 mm sample.
Find additional tips, troubleshooting help, and resources within our Real-Time PCR and Digital PCR Applications Support Center.