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View additional product information for PureLink™ Microbiome DNA Purification Kit - FAQs (A29790)
13 product FAQs found
The swab should be removed after incubation, prior to bead beating.
Yes, the elution buffer in the PureLink Microbiome DNA Purification Kit contains EDTA. An alternative that does not contain EDTA is Tris Buffer (10 mM Tris-HCl, pH 8.0-9.0). Eluting in water is possible, but the low pH of water could lead to reduced stability and less accurate OD readings.
For some samples, including soil and stool samples, it can be difficult to withdraw 400 µL supernatant while avoiding debris. If less than 250 µL of supernatant is transferred, add S1-Lysis buffer to bring the volume to 400 µL, before adding S3-Cleanup Buffer.
Low yield could be caused by inefficient lysis and/or low levels of DNA in the sample. Please try heating samples at 95 degrees C for 5-10 minutes instead of 65 degrees C for 10 minutes after adding S2-Lysis Enhancer. Bead beat for a longer time or at a higher power setting. You can also try to perform the experiment with more starting material, but do not exceed what we specify in the protocol. However for some challenging samples, too much starting material can also result in low yield. In this case, please try less starting material and/or increase the volume of S1-Lysis Buffer.
There are several options to rescue your DNA obtain good PCR results including diluting your DNA sample, using specific enzymes, and/or using an additive. The methods are described below:
- Dilute your DNA sample 10-100 times prior to PCR which will dilute out the contaminants while the PCR signal in many cases will remain strong.
- Use “tough” enzymes that were specifically designed and optimized to be effective in harsh environments, and work in the presence of high levels of inhibitors, for instance, TaqPath qPCR Master Mix, CG Reagent.
- Use a BSA additive for the PCR reaction. In most cases, if used at appropriate concentration, BSA can rescue your sample.
You can also try repeating the purification with less starting material or increased volume of S1-Lysis buffer. Additionally, after the addition of the S3- Cleanup buffer, you can try incubating the sample for 10 minutes on ice before centrifugation.
For highly pure samples, both fluorometers (such as the Qubit Flourometric Fluorometer) and spectrophotometers (such as NanoDrop One Spectrophotometer) are successfully utilized and give very similar readings. However, when dealing with DNA or RNA samples that have high levels of contaminants, such as salts or protein, the Qubit Fluorometer provides a more accurate reading, as this instrument uses dyes that are very specific to the analyte of interest. We have seen some challenging microbiome and FFPE samples where the Qubit Fluorometer readings were up to 2 times lower compared to the NanoDrop spectrophotometer, which is overestimating DNA concentrations. More and more laboratories are use both instruments side by side, as they provide complimentary information: the Qubit fluorometer is more accurate and specific in terms of determining nucleic acid concentration, while the NanoDrop spectrophotometer provides a valuable information regarding the purity (ie., A260/280, A260/230).
We tested 3 liquid versions of transport media: Amies, Stuart, and Cary Blair, with all three being compatible with the PureLink Microbiome DNA Purification kit.
Yes, the PureLink Microbiome DNA Purification Kit works for swabs samples. However this kit does not come with swabs. You will find some suggested swab part numbers that we have tested with this kit in swab protocol on the product page. (http://www.thermofisher.com/order/catalog/product/A29790).
The PureLink Microbiome DNA Purification Kit was developed to be used with a wide range of sample types including stool, soil, urine, saliva, swabs, and other samples. In addition to testing bacterial samples, we have also tested fungi samples. Specialized beads in the kit efficiently break open cell walls and membranes with the help of heat and chemical lysis. This triple lysis approach works great for tough-to-lyse species such as fungi. Additionally, the stool-derived inhibitors are rapidly eliminated with a novel clean up buffer, resulting in DNA that is compatible with any type of downstream analysis.
Yes, when using microbial DNA protocol with PureLink Microbiome DNA Purification Kit, you will get both host and microbial DNA. The bulk of DNA in human stool is of microbial origin. Depending on the sample, we roughly estimate that approximately 10-20% of the total DNA isolated is host DNA while the remaining 80-90% is microbial DNA.
We simplified the protocol for those scientists who are only interested in human DNA isolation from stool, as mammalian cells are easy to lyse and DNA isolation can be performed faster. Microbes have thicker cell membranes of a different composition, and are generally more difficult to lyse. Thus, separate protocols, with additional steps to ensure efficient disruption for isolation of microbial DNA, were developed.
If fresh stool, body fluids, swabs, and other samples cannot be processed, freezing the samples at -20 degrees C or -80 degrees C is the best way to preserve them. We recommend keeping freeze/thaw cycles to a minimum. For many samples, storage at 4 degrees C for up to one week is also fine. Please note, use of commercially available DNA stabilizing reagents can negatively impact the downstream nucleic acid isolation, or make the workflow more challenging or lengthy.
Yes, the PureLink Microbiome DNA Purification Kit has been successfully used for isolation of microbial DNA from human, mouse, rat, horse, cow, dog, cat, and other types of stool/feces. Despite the fact that these samples are very different in terms of consistency, presence of miscellaneous food debris, composition of the microbial community, various inhibitors present, and other variables, the kit enabled fast and efficient isolation of substantial amounts of highly clean microbial DNA.