Search
Search
View additional product information for Phusion Blood Direct PCR Kit - FAQs (F547S, F547L)
13 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.
We recommend using blood concentration up to 20%; however, we had successful amplifications with up to 40% of blood in the reaction mix. In some cases optimization may be required to achieve this.
Extensive testing has shown 3.0 mM MgCl2 to be optimal concentration for most PCR reactions using whole blood. There is a separate tube of 50 mM MgCl2 (F-510MG) included in the kit for adjusting the MgCl2 concentration (up to 4.5 mM if necessary) for reactions containing very high amounts of blood. It should be noted though that excess MgCl2 may result in spurious PCR products. If unspecific products are created with the default reaction buffer (providing 3.0 mM MgCl2), the effective MgCl2 concentration can be decreased by adding the chelating agent EDTA, which is included in the kit. Typically, adding 0.5 to 1.0 µL of 50 mM EDTA to a 20 µL reaction is sufficient to eliminate non-specific products. Note that the optimal conditions will depend on the primers, the percentage of blood in the reaction and/or the type of card used, since anticoagulants and other chemicals impregnating the cards can alter the available Mg2+ concentration.
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.
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.
No.
Phusion Blood DNA Polymerase has a strong 3'- 5' exonuclease activity which is able to cleave the mismatched base from the 3' end and continue amplification. If the mismatch is located at the 3' end and is not protected from cleavage by any modification, Phusion polymerase will cut the mismatched base.
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.