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Description
Highly efficient purification of host-cell and microbial DNA by combining fast lysis of the starting material in the presence of guanidinium thiocyanate with the speed and purity of silica-membrane column purification.
Product Highlights
Fast – streamlined protocol for extraction of host cell and microbial DNA in as little as 15 minutes
High-performance – recovery of consistently high-quality DNA, ideal for use in all downstream applications
Efficient – rapid and effective lysis of fecal samples by bead beating for excellent recovery of DNA
Flexible – reliable recovery of host and microbial DNA from human and a broad range of animal species
Product Description
The ISOLATE II Fecal DNA Kit provides a simple, efficient column-based method for the isolation of DNA from a wide variety of fecal samples, without the need for hazardous reagents such as phenol.
By combining the stringency of guanidinium-thiocyanate lysis and high-density bashing beads and the speed and ease-of-use of silica-membrane purification to remove humic acids/polyphenols, the ISOLATE II Fecal DNA Kit provides a fast method for the purification of high-quality bacterial/protist DNA from a variety of fecal samples including humans, birds, rats, mice, rabbits, cattle, etc. as well as host DNA.
The ISOLATE II Fecal DNA Kit has been designed to purify samples ideal for use in end-point PCR alongside any enzyme from the Meridian PCR portfolio, including MyTaq DNA Polymerase and in qPCR using the the SensiFAST Real-Time PCR Kits.
Applications
End-point PCR
qPCR
Southern, dot and slot blotting
Genotyping
Next Generation Sequencing
Bisulfite conversion/methylation analysis
PCR amplification of DNA extracted from mouse feces
Genomic DNA was extracted from increasing amounts of mouse feces (6 mg, 12.5 mg, 29 mg, 53.5 mg, 104 mg and 139 mg (lanes 1-6 respectively)) and mouse tail (control) using ISOLATE Fecal DNA Kit. PCR was performed using two primer sets for the Rn 18s gene (400 bp and 1 kb). The consistent yield of PCR product from all of the samples indicates efficient removal of PCR inhibitors, even with larger volumes of starting material.
Consistency of extraction of DNA from mouse feces
Genomic DNA was extracted from 2 uL of mouse fecal matter from different sources and compared to mouse tail DNA (control), using ISOLATE Fecal DNA Kit. PCR was performed using a primer sets for the Rn 18s gene (1.8 kb). The results illustrate the consistency of the yield of DNA from all of the fecal samples.
All components should be stored dry and at room temperature. When stored under the recommended conditions and handled correctly, full activity of reagents is retained until the expiry date indicated on the outer box label.
"Aim: To compare the sensitivity of two commercial faecal DNA extraction kits ...
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Rebecca Traub, Veterinary Sciences, University of Melbourne, Parkville, Australia
Aim: To compare the sensitivity of two commercial faecal DNA extraction kits for the detection and quantification of human hookworms (Ancylostoma ceylanicum, Ancylostoma duodenale and Necator americanus) eggs in faeces.
Methods: Ten individual human faecal samples positive for hookworm eggs were each extracted with two commercial kits, the Powersoil DNA Isolation Kit (MoBio) and the Isolate Faecal DNA Kit (Bioline) according to manufacturer's instructions. DNA was eluted to 100 ul for each.
DNA was subjected to multiplex Taq-man based multiplex qPCR (unpublished) targeting the internal transcribed spacer regions of the three human hookworms and an internal control (EHV4).
Results: Both kits performed equivocally in positively detecting at least one species of hookworm in all ten samples. The Isolate Faecal DNA Kit (Bioline) produced on average 0.5 Ct lower threshold cycles for samples compared to the Powersoil DNA Isolation Kit (MoBio). The time taken to extract 10 samples was significantly less for the Meridian product.
Conclusion: The Powersoil DNA Isolation Kit (MoBio) and the Isolate Faecal DNA Kit (Bioline) are comparable in terms of sensitivity for detecting and quantifying DNA of hookworms from human faecal samples. The Meridian kit was less labour intensive compared to the Mo Bio kit and is more competitively priced.
Rebecca Traub, Veterinary Sciences, University of Melbourne, Parkville, Australia
The columns supplied with the ISOLATE II kits may appear similar, but each type has been optimized to work within the buffer system supplied with the corresponding kit. The swapping of columns (or buffers) between kits may lead to no recovery of nucleic acid whatsoever, or at the very least severely impaired purification.
Interruption of the DNA/RNA extraction process is possible after the sample lysis only. It is possible to homogenize and lyse the samples and to store them in the freezer until use for RNA extraction. RNA clean up with a column cannot be interrupted and we recommend to avoid delays during the column purification process. If a delay is unavoidable the columns should be stored on ice.
The elution buffer is ideal for applications such as restriction enzyme digestion, sequencing and PCR. It is possible to use DNase-free water for elution, but you should expect a slightly lower yield.
Bacterial spores are effectively lysed with the bead lysis step. The use of high-speed disruptor devices and an optimization of sample incubation time are recommended.
