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Can I use PCRBIO Rapid Extract PCR Kit to extract and amplify DNA from a few dozen cells on filter paper?
Our PCR mixes work reliably with 10 or more copies of template per reaction but not lower than that. Keep the extracted DNA in the lowest volume possible and don’t dilute further after inactivating the protease. We advise setting up the assay with more cells and once the conditions are established, scaling it down from there.
Can PCRBIO Rapid Extract PCR Kit be used to extract and amplify DNA from a single colony or from a bacterial cell lysate?
Yes, it can be used to amplify DNA from bacterial colonies or bacterial cell lysate however, it’s not necessary to use the Rapid Extract PCR Kit in order to perform routine colony PCR. If you’re working from bacterial colonies use a sterile tip to pick a colony and resuspend into the 50µl PCR reaction. If working from liquid culture add 5µl of overnight culture to the final mix. Follow the general protocol and increase the initial denaturation time to 10 min at 95°C.
For qPCR, it may be important to extract DNA with the PCRBIO Rapid Extract Lysis Kit to decrease background noise and control the amount of DNA added to the reaction.
We recommend storing the extracted DNA at -20°C in the short term but for long term storage, we recommend purifying the DNA and re-suspending it in a standard buffer.
The extracted DNA should be suitable for manufacturer’s PCR kit. A version of the PCRBIO Rapid Extract PCR Kit is available without the PCRBIO HS Taq Mix Red: PCRBIO Rapid Extract Lysis kit (PB15.11).
Protease is a very active enzyme, and if it isn’t properly deactivated through the heat denaturation step, it could quickly degrade the polymerase during the PCR step.
200 mM DTT should be added to the feather and left to incubate overnight in order to break up the keratin.
5x PCRBIO Rapid Extract Buffer A (Lysis buffer), 10x PCRBIO Rapid Extract Buffer B (Protease buffer) and 2x PCRBIO HS Taq Mix Red.
This non-inhibitory dye, added to enable direct gel loading, runs at a similar rate to 200-300 bp DNA fragments on a 1% agarose gel and at 50-100 bp on a 2% agarose gel. You may notice a shift in this apparent molecular weight when running gels of different agarose content.
PCRBIO Rapid Extract PCR Kit has the same contents as PCRBIO Rapid Extract Lysis Kit plus PCRBIO HS Taq Mix Red for subsequent PCR amplification.
For Formalin-Fixed Paraffin-Embedded (FFPE) tissue specimens, we recommend 1 – 2 mm2 of a 10 µm cross-section. If you use less than that, you may not get enough DNA for PCR.
The PCRBIO HS Taq Mix Red contains a red dye for loading as well as tracking DNA during electrophoresis. In a 2% agarose TAE gel the dye migrates at a rate equivalent to DNA the size of 350bp whilst in a 1% agarose TAE gel the dye migration rate is equivalent to DNA the size of 600bp.
Rapid Extract PCR Kit was tested with the following list of tissues
- Mammalian cell cultures
- Animal tissues from a variety of organisms (Fish, lobster, Mice tails / ears, Zebrafish, Nematodes, Kidney, Hair Follicle)
- Insects like Drosophila, mosquitos, beetle (legs, wings)
- Buccal swabs
- Gram positive bacteria
- Fungal Mycelia
- Stool samples
- Fish Scale clips
The following tissues can require a pre-wash step before starting the PCRBIO Rapid Extract protocol2
- Yeast2,3 (pre-wash or digestion with zymolase to obtain spheroblasts)
- Feather4 (requires modification of the protocol see below )
- Stool sample5 (requires dilution series to get rid of inhibitors)
If you have a small amount of starting material, we recommend keeping the extracted DNA in the lowest volume possible and to avoid any further dilutions after inactivating the protease.
1 Batubara, A. et al. 2016. Study of BMP15 gene polymorphism in Boer, Kacang, and Boerka goats. Jurnal Ilmu Ternak Dan Veteriner 21(4): 224-230. DOI: http://dx.doi.org/10.14334/jitv.v21i4.1636 (2016).
2 Inglis, P.W. et al. 2018. Fast and inexpensive protocols for consistent extraction of high quality DNA and RNA from challenging plant and fungal samples for high throughput SNP genotyping and sequencing Applications. PLOS ONE | https://doi.org/10.1371/journal.pone.0206085 October 18.
3 Suzuki, T. & Iwahashi, Y. 2013. RNA preparation of Saccharomyces cerevisiae using the digestion method may give misleading results. Appl Biochem Biotechnol 169:1620–1632
4 Bello, N. et al. 2001. Isolation of genomic DNA from feathers. J Vet Diagn Invest 13:162–164
5 Nantavisai, K. et al. 2007. Evaluation of the sensitivities of DNA extraction and PCR methods for detection of Giardia duodenalisin stool specimens. J. Clinical Microbiology. 45: 581–583 doi:10.1128/JCM.01823-06
Changes in the colouration of the supernatant could be due to either pigments being released from the tissue into the supernatant or a change of pH. Changes in the supernatant colouration don’t necessarily affect the PCR reaction.
Would the PCR reaction work if my sample has a high level of inhibitors (like in blood and stool samples)?
PCRBIO Rapid Extract PCR Kit can be used with human blood however, blood components may inhibit the PCR reaction. Therefore, it’s a good idea to perform a serial dilution of the extracted DNA in order to find the optimal template concentration for the PCR amplification.
If the amplification is less than optimal, we recommend performing an isopropanol/acetate precipitation of the DNA prior to the PCR reaction.