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Plant Ex-Amp PCR Kit (with Kodaq MasterMix)
Plant Ex-Amp PCR Kit (with Kodaq MasterMix)
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Plant Ex-Amp PCR Kit (With Kodaq MasterMix) BHR10900140
Plant Ex-Amp PCR Kit offers the ultimate convenience for plant PCR. The hassle-free gDNA extraction process eliminates the conventional freezing of plant tissues with liquid nitrogen, mechanical disruption, organic extraction, column DNA purification, or alcohol precipitation. It only takes 15 easy minutes to “vortex-boil-vortex” a small piece of plant sample in proprietary extraction buffer to obtain PCR-ready template. The kit comes with sophisticated Kodaq 2X PCR Mastermix with dye; the mastermix is not only for un-beatable robustness and extreme fidelity, but also for a very streamlined and efficient PCR setup. Plant Ex-Amp PCR Kit is also very effective against some commonly known difficult samples such as pine-tree-like samples. The extracted DNA solution is stable to be stored at -20°C for at least one month.
A Review of Plant Ex-Amp Extraction Buffer Set
Introduction: The Plant Ex-Amp Extraction Buffer Set, along with two competitors’ one-step DNA extraction systems, was tested for its ability to effectively produce DNA template material for the amplification of a molecular marker in freshwater chlorophytes (green algae).
Background: Due to their great diversity and oftentimes indistinguishable morphology, algae have traditionally been very difficult to describe and classify. It is estimated that up to a million or more species of algae exist, only a fraction of which has currently been discovered and described. A major area of algal study has thus focused on the identification of DNA barcodes – easily amplifiable regions of DNA that are universally present within a certain taxonomic division and have the right amount of variability to distinguish species – which may help with the identification or discovery of species of algae. Extensive study has been conducted for one of the most well- known groups of algae, the freshwater-inhabiting chlorophytes or green algae, in an attempt to find the best barcode region. One major region – the ITS, or internal transcribed spacer portion of the ribosomal RNA genes – has been identified as a potentially suitable molecular barcode for most green algae, following its successful use in diatoms, another lineage of algae.
One major difficulty in this line of research, however, is the extraction of DNA from algae for amplification and eventual sequencing. A variety of methods have been tested and used, but they all have appreciable drawbacks. A “manual” system for DNA extraction, involving cellular lysis with a detergent such as CTAB followed by chloroform extraction and ethanol precipitation, is very commonly used, but it requires lots of consumables and is very time-consuming. Spin-column based extraction methods, usually produced in kits for utilization, have been more recently utilized; however, while throughput is increased, the procedure still remains difficult and expensive. The most recent development in DNA template preparation has been the advent of single-step buffer systems that quickly and easily extract DNA from cells and yield a buffer in the same volume, without multiple pipetting, transferring, centrifuging, or eluting steps. The only major drawback of such methods is that they normally necessitate the use of a specially-engineered robust enzyme that can tolerate inhibitors which might be present in the unpurified buffer. Therefore, for the best application of such buffer systems, it is essential that they are verified to produce a high-quality template that will not inhibit the reaction of any polymerase chosen for amplification.
Methods: Two strains of freshwater chlorophytes, named JIAC3 and JIAC6, were isolated from wild-collected water samples in the Sacramento area. Light microscopy techniques were able to identify JIAC3 as a species of Eudorina, and JIAC6 as a species of Ankistrodesmus. Clonal cultures were started from single cells isolated with micropipettes; JIAC3 was maintained in a liquid medium consisting of a soil-water and fishmeal extract, and JIAC6 was maintained on agar slants with a commercial mineral-based algal medium.
The primers used in this experiment (forward [ITS1] 5’ GGTTTCAAAGCTYTWCGTGC 3’ ; reverse [ITS4] 5’ GTAAATACCACGGCTACGRTCTT 3’) were those first described by White et al. (1990). Three buffer systems (including the E.Z.N.A. Plant Direct PCR Kit) were used to extract
DNA from the algae and prepare templates. The algae from each culture was harvested and centrifuged down at 10,000 rpm x 2 minutes, and 5 µL of concentrated algal biomass was used as starting material. Manufacturer’s instructions were followed for the preparation of each buffer, but the volumes of each component were appropriately scaled up or down to yield a final template volume of 45 µL (40 µL of buffer ingredients + 5 µL algal biomass).
PCRs were set up in 25 µL volumes. For amplification, FlashTaq HotStart polymerase (Empirical Bioscience), a standard chemically-based hot-start Taq polymerase with no additional modifications for increased robustness, was employed. Each reaction consisted of 12.5 µL FlashTaq master mix,
0.4 µM each primer, 0.5 µL template, and water to 25 µL. The PCR programs used were: 1 cycle of 94C for 3 minutes; 35 cycles of 94C for 30 seconds, annealing temperature specific to each primer set for 20 seconds, and 72C for the appropriate duration for each primer set; followed by a final extension cycle of 72C for 7 minutes and an infinite hold at 4C. PCR was performed with a GeneAmp 9600 (Perkin Elmer), and products were run on a 1.2% agarose gel with TAE buffer using the MyGel mini eletctrophoresis system and visualized with SmartGlow safe stain and the SmartDoc 2.0 visualization system (Accuris Instruments).
Results and Discussion:
Figure 1: Visualized gels of PCR products amplified using one of three single-step DNA extraction buffer systems. For each pair of reactions, the left one is amplified from JIAC6, and the right one from JIAC3. The molecular-weight marker used is the 100 bp DNA Ladder H3 (Caisson Labs).
