When using the PureLink Microbiome DNA Purification Kit with soil samples, I am getting less than 400 µL supernatant after lysis. Can you provide me with any suggestions?
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.
I am getting low yield using PureLink Microbiome DNA Purification Kit. How can I improve the yield?
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 inhibitors in the isolated DNA from environmental samples using PureLink Microbiome DNA Purification Kit. Is there a way to rescue it and obtain good PCR results?
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.
Which instrument is better for quantitation of purified DNA isolated using the PureLink Microbiome DNA Purification Kit?
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).
What transport media is compatible with PureLink Microbiome DNA Purification Kit?
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.
