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Frequently asked questions (FAQ) about Direct PCR
Direct PCR in general
Q 01: Are there any restrictions to the size of the amplicon in Direct PCR?
The shorter the amplicon, the easier the Direct PCR protocol. 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 blood samples, a 3.2 kb fragment from mouse ear tissue and a 300 bp fragment from FFPE tissues. Please see the individual protocols and experimental data on the Direct PCR page.
Q 02: When do I need to use DNARelease™ Additive?
DNARelease Additive is especially important in Direct PCR from mouse samples. In the Direct PCR protocol it is added to the loading buffer for gel electrophoresis. This will prevent the PCR products from getting trapped in the agarose gel wells. In the dilution protocol for mouse samples, DNARelease is added to the TE buffer in order to aid releasing the DNA from the sample. See Phire® Animal Tissue Direct PCR Kit.
If you notice that the DNA sample is stuck in the wells with other sample material, adding DNARelease to the loading buffer may be helpful.
Q 03: How should I store the PCR products amplified directly from the sample?
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°C.
Q 04: Can I purchase Dilution Buffer separately?
The Dilution Buffer is included in Phire® Plant Direct PCR Kit and Phire® Animal Tissue Direct PCR Kit. The buffer cannot be purchased separately.
Blood samples
Q 01: What kind of blood samples are suitable for Thermo Scientific Phusion Blood Direct PCR Kit?
Fresh blood or blood stored at +4°C or frozen, and preserved with EDTA, citrate or heparin are all suitable for this kit, as is blood dried onto commercially available cards such as Whatman 903® and FTA® cards. Phusion Blood Direct PCR Kit is compatible with blood samples from various species. You can find more details on the Phusion Blood Direct PCR Kit product page.
Q 02: Can I use Phusion Blood Direct PCR Kit for allele-specific PCR directly from blood (e.g. HLA-typing)?
It depends on the primers used. If the mismatch is located at the 3’ end and is not protected from cleavage by any modification, the answer is 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.
Q 03: I’m trying to do restriction digestion to the PCR product amplified with Phusion Blood Direct PCR Kit without purifying the PCR product. The digestion is not working well. What could I do to improve the efficiency of the digestion?
There are two things that may cause the inefficient digestion. Either the restriction enzyme used is not fully active in Phusion Blood PCR buffer or it may be sensitive to blood derived inhibitors present in the reaction mixture. Usually diluting the PCR product 1:2 in H2O helps to dilute the buffer and/or inhibitors in the reaction, allowing the restriction enzyme to perform optimally. If the PCR yield is high, it is possible to dilute the PCR product even more. If the dilution doesn’t help, it is recommended to purify the PCR product before the digestion. However, many restriction enzymes perform well in the PCR product amplified directly from blood. See the application protocol for PCR-RFLP directly from blood.
Q 04: Can I do real-time qPCR with Phusion Blood Direct PCR Kit if I add SYBR Green to the reaction?
Phusion Blood Direct PCR Kit is optimized only for end-point PCR, not for quantitative analysis. The blood present in the PCR reaction may interfere with fluorescence detection and decrease the amplification efficiency.
Q 05: Does Phusion Blood Direct PCR Kit have an IVD-CE mark?
No.
Mouse tissue
Q 01: Can I use other parts of mouse than ear or tail when doing direct PCR from mouse tissue?
Our protocols for Direct PCR from mouse tissue have been optimized for ear and tail tissues. However, it may be possible to use other parts of mice as well, but it is very important to only use a tiny section of tissue in the Direct PCR reaction. Harris’ Uni-Core puncher is an ideal tool for sample preparation.
Q 02: I’m testing your Direct PCR protocol using mouse tissue as sample material. I can’t see a PCR product in the agarose gel, but there is a big junk of DNA stuck in the wells. What can I do?
When amplifying DNA directly from mouse tissue, it is important to add DNARelease Additive to the loading buffer when analyzing PCR products on gel. This is because cell debris present in these PCR products can cause DNA fragments to get trapped in the agarose gel wells. DNARelease Additive eliminates this problem when included in the gel loading buffer. It may also be helpful to decrease the amount of tissue in each reaction.
Plant material
Q 01: Which plants are suitable for Phire® Plant Direct PCR Kit?
Numerous plant species have been tested with Finnzymes' Phire Plant Direct PCR Kit. Please see the updated list at the Direct PCR page.
Q 02: Which parts of the plant can I use with the Phire Plant Direct PCR Kit?
This kit's protocols have been optimized for plant leaves, seeds and flowers. Other parts of the plant such as fruits and roots may also be compatible, but should be tested for the species of interest.
Q 03: Can I use frozen leaves?
Fresh plant leaves are recommended, but frozen and dried leaves can also be used. We have succeeded in amplifying DNA directly from oak leaves stored at -20°C for 10 years.
Q 04: How long can I store the DNA sample in Dilution Buffer?
The storage time depends on the plant material used. Usually the DNA sample is stable at least for few months in Dilution Buffer at -20°C. However, good results have been obtained for samples stored up to 2 years under these conditions.
Q 05: Can I use the kit for pathogen DNA detection?
Yes, some researchers have been using the kit for pathogen detection. It is important to keep in mind that the sensitivity is very critical in these applications and the sample amount may need to be titrated.
Q 06: What if I want to clone or sequence the PCR product? Can I use the kit with Phusion?
Yes. Due to the extremely high inhibitor resistance of Phire and Phusion both DNA polymerases can be used with plant material. However, it is good to keep in mind that kit is optimized for Phire. Therefore the results may be less optimal with Phusion.
Q 07: Can I get the kit without the puncher or the mat?
No. The kit includes reagents and tools for both protocols. The puncher and the mat are recommended for sampling with the direct protocol.
FFPE tissue
Q 01: Which FFPE sample types are suitable for Direct PCR from FFPE tissues?
FFPE tissue sections, tissue blocks and microscope slides can all be used in sample preparation.
Q 02: How old FFPE tissue samples can be used for Direct PCR?
The success depends on the integrity of DNA, which is typically lower in older FFPE tissue samples. Old samples can be used for sample preparation, but the yields obtained are usually lower. The length of the amplifiable DNA fragment can be limited as well.
Q 03: I’m doing PCR directly from Proteinase K treated FFPE tissue. Sometimes I don’t get a PCR product or the product yield is very low. Can you offer any suggestions?
There are a few possible reasons for low yield. If the PCR reaction conditions are optimal (positive control from purified DNA is amplified well), a typical reason for low yield is non-optimal template amount. Usually there is too much DNA or too many inhibitors in the sample. Diluting the template 1:10 or 1:100 in 1x Phusion HF Reaction Buffer helps to overcome this problem. In some cases it is also possible that there isn’t enough template DNA present. In that case it can be helpful to determine the optimal template amount by titration. When analyzing FFPE tissue samples, it is also good to keep in mind that DNA integrity from sample to sample can vary a lot, and in some samples DNA may be too degraded for PCR analysis.
