With the Powersoil extraction kit we get high DNA concentration readings and high contaminant ratios by UV-Vis spectrophotometry. However we can not visualize the DNA in agarose gels (1% or 0.7%).
Hello, please see attachment, here the well known universal DNA extraction method. You can use it for many approaches. If necessary I can send procedures for extraction.
Hello, please see attachment, here the well known universal DNA extraction method. You can use it for many approaches. If necessary I can send procedures for extraction.
I've had great results using a modified version of the Powersoil extraction kit to increase my yield and purity (from a sandy loam). A research specialist from MoBio sent it to me. It involves a phenol-chloroform-isoamyl treatment and additional wash steps. Just keep in mind that this will extract both DNA and RNA. Some things to consider; the lysis step, fragmented DNA can contribute to concentration readings but will be difficult to visualize as gel bands. Also, contamination which can contribute to pseduo high concentrations. Once this is cleaned up it should be easier to assess the actual yield. Good luck!
Thank you very much to both of you for sharing your procedures.
We have tried the modified version of the Powersoil DNA extraction Lauren Hale suggested on our sand samples. However we can not visualize the DNA as gel bands.
Lauren, how did you purify your samples? Also, did you go ahead and do PCR of the purified DNA even without being able to visualize the DNA on the gels?
Using the CPI modified version of the kit my DNA yields were typically 20-30 ng/ ul and 5-10 ul of these appeared as fuzzy, faint bands at about 10 kb. I used this DNA directly and ran both PCR and qPCR, both worked well. The MoBio rep also suggested increasing the starting material to 0.5- 1 g if I was having yield issues. They also have a different kit for samples with low quantities of DNA which requests about 10 g of starting material. How are you lysing the cells initially? I did not have the MoBio vortex bed adapter so I used a Fast Prep bead beater and had better luck with my yields. Previously, I had been taping the tubes to a flatbed vortexer and was recovering little to no DNA. Best of luck to you!
I think we need to buy a bead beater. I will also try using the DNA directly to run PCR.
Thank you Lauren!
Using a Bead Beater and the CPI modified version of the PowerSoil extraction kit from MoBio has made all the difference. Our DNA yields are now typically 10-50 ng/ ul sometimes more. PCR is working and the bands on the agarose gel are sharper.
hello..i would ike to add my question to this. i am currently using the MoBio powersoil kit for DNA isolation but i am not getting enough DNA. i got faint bands upon agarose gel run. i still amplified my samples but the pcr failed a multiple no. of times. my boss told me to try changing the pcr mixture concentrations. what should i do to get better yields using the Powersoil kit. please help.
Try the new PureLink Microbiome DNA Purification Kit – it has good protocols for stool and soil, and other microbiome samples http://www.thermofisher.com/order/catalog/product/A29790?ICID=search-product
- Badges
- Science method
- Similar topics
- Methodology
- Absorption Spectroscopy
- UV-Visible Spectroscopy