Frequently Asked Questions

MO BIO Frequently Asked Questions

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Arrow FAW DNA RNA Isolation questionsSoil

Q. What is the average DNA yield from soil?

A. The DNA yield extracted from soil is influenced by many factors including texture, pH, moisture, carbon content, and collection depth, making it pretty hard to predict. Compost and soils from the uppermost layers of the ground will have the highest yields. Clay and sediment usually have the lowest yields. In our labs, loamy soils typically yield between 3-6 micrograms DNA per 0.25 g of soil. This equates to between 12-24 micrograms per gram of soil. For our richest soils, typical yields are 10 micrograms of DNA per 0.25 grams of soil. With clay, sand, sediments, and some agricultural soils, yields can be as low as 250-500 nanograms for 0.25 grams of soil.

Q. How much RNA should I expect to extract from soil?

A. Typical RNA yields for soil are much lower than for DNA, usually 10-20% of what you would expect for DNA. See average yields for DNA above.

Q. What is the difference between the PowerSoil® and the PowerLyzer™ PowerSoil® Kit?

A. The difference between the PowerSoil® DNA Isolation Kit (catalog # 12888) and the PowerLyzer™ PowerSoil® DNA Isolation Kit (catalog # 12855) is in the bead tubes. The PowerSoil® Kit has bead tubes with 0.7 mm garnet irregular beads and the PowerLyzer™ PowerSoil® Kit has bead tubes containing 0.1 mm smooth glass beads. Which one you will want to use depends on the soil type, the homogenization speed, and homogenization equipment. It is best to analyze your soil the first time with different beads and bead-beating speeds to see which gives the best yield with minimal DNA shearing. However, as a general rule, you will see the greatest differences in DNA yield and integrity when using a desktop homogenizer (like the PowerLyzer™ 24 High powered Benchtop Homogenizer) at speeds over 3200 rpm, with glass beads giving higher yields with less shearing. You will likely see less differences between the two types of beads with a Vortex Adaptor. We always suggest that DNA samples be analyzed on both an electrophoresis gel and with a spectrophotometer. A higher reading on the spectrophotometer does not necessarily equate to a better result as sheared DNA will give an artificially high 260 nm reading. See Application Note and Poster.

Q. What homogenization protocol should I use for my soil?

A. Again, the best homogenization protocol is highly soil-type dependent. It is best to analyze your soil the first time with different beads and bead-beating speeds to see which give the best yield with minimal DNA shearing. However, as a general rule, start with lower speeds (2500 RPM for 45 seconds on the PowerLyzer™ 24 High powered Benchtop Homogenizer) when working with loose, granular and high organic soils and use higher speeds (4000 RPM for 45 seconds on the PowerLyzer™) with clay soils. Bead tubes with glass beads tend to give higher yields with less shearing at the higher (>3500) speeds. We always suggest that DNA samples be analyzed on both an electrophoresis gel and with a spectrophotometer. A higher reading on the spec. does not necessarily mean a better result, as sheared DNA will give an artificially high 260 nm reading. For more detailed information see Application Note.

Q. How can I get the highest DNA yield from my soil?

A. To increase the DNA yield in your soil we recommend using the PowerLyzer™ PowerSoil® DNA Isolation Kit (cat# 12855-50) with a high powered bead beater like the PowerLyzer™ 24 High powered Benchtop Homogenizer (cat# 13155). Experiments with a variety of soil types (see Comparison of Microbial Populations Isolated from a Variety of Soils using Different Homogenization Methods During DNA Extraction) have shown that a glass bead matrix (0.1 mm glass) coupled with the PowerLyzer™ 24 High powered Benchtop Homogenizer give the highest DNA yield for most soil types, but most especially for soils high in carbon content. You can also potentially increase yields by increasing the amount of starting material up to 500 mg. However, with larger amounts of starting material there is the concern that there won't be enough liquid in the bead tube to homogenize the sample effectively. If necessary add 100-200 ul more bead solution to the tubes before homogenization. Extra bead solution is sold separately (12855-50-BS). For soils high in water content you can try centrifuging the sample and removing the liquid, allowing you to add more dry weight. Be sure that when removing the supernatant after bead beating there is at least 400 ul and no more than 600 ul of liquid. Also see Application Notes.

Q. I want to look at fungal DNA in my soil. How can I make sure that the spores are lysed open?

A. To increase the lysis of hard to break microbes like spores and gram positive bacteria try heating your sample in the lysis buffer in the bead tube at 65 °C for 10 minutes immediately before bead beating. Note that heating the sample will decrease the amount of RNA.

Q. What is the difference between the PowerFecal™ DNA Isolation Kit and the PowerSoil® DNA Isolation Kit?

A. The PowerSoil® DNA Isolation Kit (catalog # 12888-50) and the PowerFecal™ DNA Isolation Kit (catalog # 12830-50) contain identical chemistry and both use 0.7 mm garnet bead tubes. However, the PowerSoil® DNA Bead Tubes come pre-loaded with Bead Solution where the PowerFecal™ DNA Isolation Kit contains a separate bottle of Bead Solution that is added by the end user. In addition, the PowerFecal™ DNA Isolation Kit incorporates a 65 degrees C heating step into the protocol and some additional tips for improving the DNA yield from fecal samples.

Q. Can the PowerFecal™ DNA Isolation Kit and the PowerSoil® DNA Isolation Kit be used interchangeably?

A. Yes, the PowerSoil® DNA Isolation Kit (catalog # 12888-50) and the PowerFecal™ DNA Isolation Kit (catalog # 12830-50) contain identical chemistry and can be used interchangeably for both soil and fecal samples. The Bead Tubes in the PowerSoil® DNA Isolation Kit come pre-loaded with Bead Solution whereas the PowerFecal™ DNA Isolation Kit contains a separate bottle of Bead Solution that is added by the end user. The PowerFecal™ DNA Isolation Kit protocol contains an additional heating step immediately before bead beating. (65 degrees C for 10 minutes) We do not recommend this heating step for soil samples high in humic acids. However, we do recommend it for fecal samples.

Q. Can I increase the amount of my soil sample?

A. The PowerSoil® DNA Isolation kits were optimized for 250 mg of starting sample for most sample types. However, if your sample type has a very low moisture content you can potentially increase the amount of starting material up to 500 mg. With larger amounts of starting material there is the concern that there won't be enough liquid in the bead tube to homogenize the large sample. Be sure that when removing the supernatant after bead beating there is at least 400 ul of liquid. If necessary add 100-200 ul more bead solution to the tubes before homogenization. Extra bead solution is sold separately (12855-50-BS).

Q. I am using a PowerSoil® DNA Isolation Kit with a low biomass sandy soil. How can I increase my DNA yield?

A. For soils low in biomass and also low in humic acids you may simplify the Inhibitor Removal Technology® (IRT) steps. When you get to the solution C2 step, reduce it to only 100 ul. Next add the same amount of Solution C3 (100 ul) at the same time instead of waiting until the next step. Incubate at 4 °C for 5 minutes and proceed directly to the step after C3. This saves handling that might reduce your DNA yield. Also, with the reduced volume at these steps, you can try taking all 600 ul after the homogenization step, follow with Solution C2 and Solution C3 added at the same time, and proceed to step Solution C4.

Q. What is the DNA/RNA extraction efficiency for the MO BIO Soil DNA and RNA Extraction Kits?

A. People often ask us if they are getting all of the DNA from their soil, especially if they have a low yielding soil. Since every type of soil is different, with varying pH, humics, and carbon content, a good control is to spike your soil sample with a known quantity of bacteria and run it through the kit. You can start with the pellet from 1 ml of liquid culture (make sure it's fresh) of a known bacteria. Here's a good rule of thumb for E. coli: 1 ml of overnight culture contains 10^9 cells or approximately 5 micrograms of DNA. Spin down the cells from a 1 ml culture into a pellet and then resuspended in 100 uL saline. Add it to the soil sample and let incubate for at least 10 minutes before doing the prep.

Q. I am using the PowerSoil® DNA Isolation kit. After adding Solution C2 and centrifuging, the instructions say to transfer up to 600 ul of supernatant to a clean 2 ml collection tube. However, I have more than this. Is it okay to take it all? I don't want to lose any sample.

A. It is okay to take more than 600 ul. However, you do not want to take more than 750 ul after the C3 step as the proportions to Solution C4 are critical. If you find that you have too much supernatant after adding C3 you may want to reduce the amount of bead solution in the bead tube by 200 ul at the beginning of the protocol. Alternatively, if you have a very wet soil, you can spin the soil and remove some of the liquid before adding it to the bead tube.

Q. I am seeing some RNA degradation with the MO BIO PowerSoil® Total RNA kit. How can I reduce it?

A. We suggest that you add the phenol solution before starting the homogenization, at the very beginning when you add the Bead Solution, SR1 and SR2 to the tube. Do no wait until five minutes into bead beating as stated in the protocol. Some samples are especially prone to degradation because of a high level of dead bacteria and RNases.

Q. Can I use RNALater® or RNAProtect® to preserve nucleic acids in my soil?

A. No! These get stuck in the soil and cannot easily be washed out. The remaining solution will make DNA and RNA extraction virtually impossible. Instead, we suggest that you use the MO BIO LifeGuard Soil Preservation Solution. It will preserve nucleic acids in soil during storage and transport for up to 30 days at -20 Celcius. See Application Note.

Q. The PowerSoil kit states that it only isolates DNA, but I don't see an RNAse step. Is there one? Where should I add RNase?

A. This kit does not contain RNAse because low molecular weight nucleic acids will not bind to the spin column. The RNA washes through. Typically there is no visible RNA when the eluted DNA is run on a gel. However, it is possible that there could be very small amounts. If you need to ensure there is absolutely no RNA you can add 1 ul of RNase to the Bead Tube at the beginning of the protocol. 

Q. I don't have a high powered bead beater and I have a very low yielding soil. Is there anything I can do to increase the yields?

A. Yes, we do have an alternative protocol for improving DNA yields from low biomass samples using additional phenol:chloroform:isoamyl alcohol, pH 7-8 and 100% ethanol. For instructions please contact technical support.

Arrow FAW DNA RNA Isolation questionsMicrobial

Q. Can the UltraClean® Microbial DNA isolation Kit be used to isolate genomic DNA as well as plasmid DNA?

A. The UltraClean® Microbial DNA isolation Kit has been optimized as a genomic DNA isolation kit but it will also co-isolate plasmid DNA. Typically, smaller species of DNA and RNA (< 3 kb) do not bind with high efficiency. However, it is possible to recover smaller molecular weight nucleic acids by altering a step. If you want to increase the recovery of small DNA, you should increase the amount of MD3 that you use for binding the DNA to the silica column. The usual protocol uses 900 ul of MD3 for 450 ul of microbial lysate. By adding a third more volume of MD3, 1.2 ml, you will increase the recovery of smaller nucleic acids. Run the product on a gel with a ladder to confirm.

Q. Is RNA contamination a concern when using th UltraClean® Microbial DNA Isolation Kit? Which step in the protocol eliminates the presence of RNA from the genomic DNA?

A. The UltraClean® Microbial DNA Isolation Kit selectively binds DNA during the binding step. However, none of the reagents contain RNase so it is possible that there is some carry through. In most cases, contaminating RNA is not a problem. If complete RNA removal is critical, RNase A can be added to the bead solution in the first step of the protocol.

Q. Can your UltraClean® Microbial DNA Isolation Kits be used to isolate DNA from both gram negative and gram positive microbial cultures?

A. Yes, both the UltraClean® Microbial and the PowerLyser UltraClean® Microbial DNA Isolation Kits will work with all types of microbes including gram negative & gram positive bacteria, fungi, and yeast cultures.

Q. I am processing a large number of samples with the UltraClean® Microbial DNA Isolation Kit. Where is a good stopping point in the protocol to take a break?

A. A good stopping point would be after the addition of Solution MD2. Samples can be stored at 4°C for a period of a few hours to overnight without any problems at this point.

Q. I have been using the UltraClean® Microbial DNA Isolation Kit and I have been having trouble with my 260/280 and 260/230 ratios. How can I improve the purity of my eluted DNA?

A. Try a second wash prior to elution using Solution MD4 or 80% ethanol. After the second wash increase the dry spin to two minutes at 13,000 x g to completely remove any residual ethanol and to dry the column prior to elution.

Q. What are some typical DNA yields using the UltraClean Microbial DNA Isolation Kit?

A. The DNA yield varies depending on the type of microbe and growing conditions. Some typical average yields from a 1.8 ml culture as measured on a Nanodrop are 14 ug for Escherichia coli, 11 ug for Enterococcus faecalis, 5 ug for Bacillus subtilis and 4ug for Candida albicans. There may be a small amount of RNA carry-through contributing to these values.

Arrow FAW DNA RNA Isolation questionsWater

Q. Should I use the PowerWater® or the RapidWater™ DNA Isolation Kit?

A. The PowerWater® DNA Isolation Kit includes our patented Inhibitor Removal Technology® (IRT) and works best for turbid water samples containing dissolved organic matter or metals. If your sample is clean ground water or from some potable water source then the RapidWater™ DNA Isolation Kit can be used and will be faster. If you are unsure which to use then go with the PowerWater® DNA Isolation Kit.

Q. Can I use my own filter membranes or do I have to order yours?

A. Our water kits will work for all types of commonly used filter membranes such as those made from polyethersulfone, cellulose acetate, nitrocellulose, and polycarbonate. As long as the filters are 47 mm in diameter they will work with the MO BIO PowerWater® and RapidWater™ DNA Isolation Kits.

Q. Can I co-isolate RNA and DNA using the PowerWater® DNA Isolation kit?

A. The PowerWater® DNA Isolation Kit is optimized for the isolation of DNA. For RNA isolation from filtered water samples we recommend the PowerWater® RNA Isolation Kit (MO BIO Cat# 14700). For co-isolation of DNA and RNA with the PowerWater® RNA Isolation Kit leave out steps 19-23 of the protocol.

Q. Can the PowerWater® DNA Isolation Kit be used for isolation of DNA from filtered liquids other than water? What about filtered air?

A. Yes, the PowerWater® DNA Isolation Kit can be used for microbes filtered from virtually any liquid or air. It has been used for urine, sewer water, dusty air, and even contaminated oil well water.

Q. Can I use other types of filter units like Millipores's Sterivex™ or SteriPak™ with the PowerWater® DNA Isolation Kit or the RapidWater DNA Isolation Kit?

A. Yes, Sterivex® and SteriPak® filter units can be used with the PowerWater® or the RapidWaters® DNA Isolation kits. Before doing so, the filter unit plastic housing must be opened, the filter membrane removed and then cut into pieces before placement into the bead tube. A kit for in-unit DNA extraction is available for Sterivex™ Filter Units (MO BIO Cat# 14600).

Q. I have some frozen water samples. Can I use the PowerWater® DNA Isolation Kit?

A. Freeze/thawing of water samples may result in microbial lysis which cannot then be filtered onto membranes without risking loss of nucleic acids. Samples in smaller aliquots (such as those concentrated via centrifugation) may be processed using other kits such as the PowerSoil® or PowerMax® DNA Isolation Kits (MO BIO cat# 12888, 12988). Otherwise, if freezing the samples is desired we recommend filtering them onto membranes first and then placing the membranes into the freezer. Many people store the filters inside the PowerWater® bead tubes when freezing so that the filter membrane can be processed directly using the kit without the need for transferring.

Q. What's the best way to preserve the DNA in my water samples before extraction?

A. The best way to store the samples is to filter your water through a membrane, place the membrane into the dry PowerWater® Bead Tube and store at -20°C. If freezing is not an option, the membrane can be dried down under vacuum and placed dry into the bead tube before storing.

Q. I have no easy way to carry my water back to the lab. Is there a way for me to collect the microbes on filters in the field?

A. Many of our customers use Sterivex Filter Units in order to filter large quantities of water (1-10 liters) in the field. To see how, watch a video demonstration by Russell Neches from the Jonathan Eisen Lab at UC Davis. For fast and easy isolation of high quality genomic DNA from Sterivex™ Filter Units, the PowerWater® Sterivex DNA Isolation Kit is ideal.

Q. Do I need a vacuum manifold for the PowerWater® Sterivex™ DNA Isolation Kit?

A. Yes, a vacuum manifold is necessary to bind the lysate to the binding columns.

Q. My water filtered Sterivex™ units have been stored with SET (Sucrose, EDTA, Tris) lysis buffer added to them. Can I still use the PowerWater® Sterivex™ DNA Isolation Kit for these samples?

A. You would need to remove the SET buffer before using the MO BIO PowerWater® Sterivex kit because the EDTA in the buffer may interfere with the kit chemistry. In addition, EDTA can lyse some of the organisms on the Sterivex membrane, especially if they have been stored frozen. DNA from lysed cells will be free-floating in the buffer. Storing the units dry and following our kit protocol gives the best yields compared to enzymatic methods. See Application Note.

Q. My water sample is not on a filter but has been condensed into a small volume of liquid. What kit should I use?

A. There are a couple of options. If you are interested in the bacterial DNA you can pellet the cells from the condensed water and use one of our microbial kits like the UltraClean® Microbial DNA Isolation Kit or the PowerLyser™ UltraClean® Microbial DNA Isolation kit. However, this would not be ideal for viral DNA as the virus will remain in the supernatant. In this case you could use the PowerWater® DNA Isolation kit without a filter. However, you may need to reduce the lysis buffer that you add to the bead tube by 200-400 microliters so that the total volume (lysis buffer+water sample) you remove from the bead tube after lysis is no more than 600-650 ul.

Q. Is there a way of isolating RNA from the Sterivex™ filters using the PowerWater® Sterivex™ DNA Isolation Kit?

A. Yes, we have an alternative protocol for isolating RNA from Sterivex filters using the PowerWater® Sterivex DNA Isolation Kit. Contact Technical Support for the protocol changes.

Q. Where can I purchase Sterivex™ water filters?

A. The Millipore Sterivex water filter units (Millipore catalog #SVGPL10RC) can be purchased directly from Millipore or from any distributor of Millipore products such as VWR.

Q. Is it possible to use the Sterivex water kit with Sterivex filter units that do not have a male Luer-Lok® outlet?

A. Yes, if you have a Sterivex filter unit with a male nipple outlet you can still use it with the kit. However, you will need another method to seal the outlet. Some customers use a bit of Hemato-Seal Capillary Tube Sealant (Fisher cat# 02-678). Another option is to cut a syringe tip cover in half and use it as a cap for the outlet.

Arrow FAW DNA RNA Isolation questionsFecal

Q. What is the difference between the PowerFecal™ DNA Isolation Kit and the PowerSoil® DNA Isolation Kit?

A. The PowerSoil® DNA Isolation Kit (catalog # 12888-50) and the PowerFecal™ DNA Isolation Kit (catalog # 12830-50) contain identical chemistry and both use 0.7 mm garnet bead tubes. However, the PowerSoil® DNA Bead Tubes come pre-loaded with Bead Solution where the PowerFecal™ DNA Isolation Kit contains a separate bottle of Bead Solution that is added by the end user. In addition, the PowerFecal™ DNA Isolation Kit incorporates a 65 degrees C heating step into the protocol and some additional tips for improving the DNA yield from fecal samples.

Q. Can the PowerFecal™ DNA Isolation Kit and the PowerSoil® DNA Isolation Kit be used interchangeably?

A. Yes, the PowerSoil® DNA Isolation Kit (catalog # 12888-50) and the PowerFecal™ DNA Isolation Kit (catalog # 12830-50) contain identical chemistry and can be used interchangeably for both soil and fecal samples. The Bead Tubes in the PowerSoil® DNA Isolation Kit come pre-loaded with Bead Solution whereas the PowerFecal™ DNA Isolation Kit contains a separate bottle of Bead Solution that is added by the end user. The PowerFecal™ DNA Isolation Kit protocol contains an additional heating step immediately before bead beating. (65 degrees C for 10 minutes) We do not recommend this heating step for soil samples high in humic acids. However, we do recommend it for fecal samples.

Q. What is the difference between the RNA PowerSoil® Total RNA Isolation Kit and the PowerMicrobiome™ RNA Isolation Kit?

A. Both of these kits can be used to extract RNA from feces or stool. The RNA PowerSoil® Total RNA Isolation Kit uses a starting sample size of 2 g, a 15 ml Bead Tube, and an ion exchange column. The PowerMicrobiome™ RNA kit uses a starting sample size of 0.25 g, a 2 ml Bead Tube, and a silica column. This makes the PowerMicrobiome™ RNA kit kit faster, easier, and cheaper to use. There are no ethanol precipitations required. However, the RNA PowerSoil® Total RNA Isolation Kit allows you to elute DNA & RNA separately from the same column using the RNA PowerSoil DNA Elution Accessory Kit. Using the PowerMicrobiome™ RNA kit you can extract both DNA and RNA but you must elute them both at the same time and then treat with DNase/RNase as desired.

Q. I would like to extract DNA from avian/bird feces. Which kit should I use for this application?

A. We would recommend either our PowerLyzer™ PowerSoil® or our PowerSoil® DNA Isolation Kits. These have our strongest inhibitor removal process. The PowerLyzer™ PowerSoil® Kit has bead tubes with 0.1 mm glass beads which can sometimes give a higher yield than the original PowerSoil® kit with 0.7 mm garnet beads. It depends on the sample consistency and types of microbes. Many of our customers working with stool and gut samples incorporate a 65C heating step for 10-15 minutes before vortexing to increase cell lysis.

Q. I have some stool samples that are already in guanidine thiocyanate (GTC) with 1% beta-2 mercaptoethanol. Can I leave out the first buffer in the bead tube and bead beat in the GTC instead?

A. No, buffers high in guanidine thiocyanate will interfere with the inhibitor removal chemistry in the kit.. It's better to pellet the sample and remove the buffer before resuspending in the bead tube solution.

Q. I want to isolate DNA and RNA from the same stool sample. What should I use?

A. There are a couple of different options for simultaneously extracting DNA & RNA from fecal samples. We have the RNA PowerSoil® Total RNA Isolation Kit (12866-25) which can be used in conjunction with a DNA Elution Accessory Kit (12867-25) to extract both DNA & RNA separately from the same column. That kit can use up to 2 grams of sample. If you can use less sample (0.25 g) the PowerMicrobiome™ RNA kit also allows you to extract both DNA and RNA but you must elute them both off the column at the same time and then treat with DNase/RNase as desired. However, the PowerMicrobiome™ RNA kit is the faster and less expensive kit to use.

Q. I am following the Human Microbiome Project Manual of Procedures and it refers to the MO BIO PowerSoil kit and some additional "MO BIO Lysis buffer." I don't see this listed on your website. What do I need?

A. The solution that the HMP refers to as the "MO BIO Lysis buffer" is our PowerSoil Bead Solution. It can be purchased in a 42 ml bottle with Catalog# 12855-50-BS

Q. How do I collect and store a fecal sample for DNA/RNA isolation?

A. The microbial community can maintain its structure quite well for up to two weeks even at room temperature. We suggest collecting samples in Bead Tubes and either storing at -20 C or shipping with standard cold packs. Once frozen avoid freeze/thaw cycles. For more detail please see our blog article The Scoop on Poop... Collection and Transport.

Arrow FAW DNA RNA Isolation questionsPlant

Q. Can I use the PowerPlant RNA Isolation Kit for isolation of microRNA and siRNA?

A.The PowerPlant RNA kit will work well for small RNAs with a minor modification. For the purification of microRNA and siRNA we suggest the addition of an extra 650 ul of 100% ethanol before the step that binds the RNA to the spin filter. The rest of the protocol proceeds as usual.

Q. Can I use the PSS (Phenolic Separation Solution) with any plant type to increase my nucleic acid yields?

A. No. Plant samples have varying amounts of phenolics. If there are a lot of phenolic compounds in the plant sample (grape leaf, strawberry leaf, tomato stem, pine needles), then these can interfere with the DNA/RNA extraction. The Phenolic Separation Solution (PSS) helps remove these phenolic compounds and increase your yield. However, if your sample does not contain high levels of phenolics (Cotton seed, grass leaf, rice leaf, mint leaf) and you utilize the PSS you can actually reduce your yields. Also be aware that phenolics are not always evenly distributed within the entire plant structure, so the need for using the PSS is contingent on the portion of plant you are working with. If you are uncertain as to the levels of phenolics in your particular sample, run a comparative assay with and without PSS.

Q. Should I heat my sample prior to bead beating as expressed in most other protocols?

A. Not necessarily. With the PowerPlant Pro chemistries we have found that not all samples should be heated prior to bead beating; yields can actually be reduced when the sample is heated. Examples of this include Cotton Leaf, Grass, Pine Needles and Grape leaves. It is suggested to perform a comparative assay with and without heat on your particular sample type to optimize the protocol.

Q. I am extracting microbial DNA from wood and roots and am concerned about polysaccharides and polyphenolics, which may potentially be present in high concentrations in these samples. What kit should I use?

A. We recommended the PowerPlant Pro Kit. The PowerPlant Pro Kit has 2.38 mm metal beads which are very effective for plant sample homogenization. It will break down hard samples like wood or roots to release microbes that may be hiding inside. But it will also release plant DNA. If the microbes you want are not inside the tissue, but on or close to the surface, you could use a 0.1 mm or 0.5 mm glass bead tube to minimize plant DNA release. For more detailed information see: Knock on Wood… and Isolate Fungal DNA!

Arrow FAW DNA RNA Isolation questionsTissue & Cells

Q.Can I use DNA extracted with the BiOstic FFPE Tissue & Cells DNA Isolation Kit for Next Generation Sequencing?

A. Yes, DNA Isolated from the BiOstic FFPE Tissue DNA Isolation Kit has been successfully used for PCR, qPCR, and Next Generation sequencing. Keep in mind that although the BiOstic FFPE Tissue DNA Isolation Kit provides effective but gentle removal of contaminants that can interfere with downstream applications, the kit will give back the same quality of DNA that goes in. If degraded DNA is in the FFPE tissue slice, then this is what will come out.

Q. Can I use the BiOstic FFPE Tissue & Cells DNA Isolation Kit to extract RNA?

A. Yes, we have an alternate protocol that can be used with the BiOstic FFPE Tissue DNA Isolation Kit that allows you to use the same kit for RNA isolation as well. You need to make the following changes: 1) A lysis buffer that protects the RNA during the heating steps is used to digest the tissue through the wax and to de-crosslink the proteins from the DNA. For this we use Solution BF2 from the PowerBiofilm DNA Kit. The catalog number is 24000-50-2. Add 200 ul of BF2 in place of FP1 and FP2. Add the 20 ul of FP3. Digest the sample for 1 hour at 60 C. 2) The temperature used to de-crosslink the protein and RNA needs to be lowered to 70 C for 30 minutes (instead of the 90 C for 60 minutes used for DNA). If the sample is only paraffin embedded, not formalin fixed, you can skip this step. 3) Add Solution FP4 and 1.5 volumes of 100% ethanol (300 ul) at step 7 instead of 200 ul of FP5. Up to 2 volumes of 100% ethanol can be added to increase the recovery of miRNA and siRNAs. 4) If only RNA is desired, incorporate the On-Spin Column DNase Kit (cat # 15100) into the protocol to remove the genomic DNA from the membrane before elution. Call technical support on how to insert these steps into the protocol.

Q. Do I need a high powered bead based homogenizer to extract RNA from tissues using the UltraClean Tissue & Cells RNA Isolation Kit?

A. The answer is no; but it's the fastest and easiest method. Other methods for homogenization include the use of liquid nitrogen and hand held rotor-stators. For RNA especially, the homogenization method must be fast enough to break down samples without overheating or degrading the RNA. High powered bead-based homogenizers can process multiple samples at once at high speed while maintaining the integrity of the RNA. . For more detailed information see the Application Note: "Isolation of RNA from Animal Tissues using the PowerLyzer™ UltraClean® Tissue & Cells RNA Isolation Kit."

Q.Can I use the UltraClean Tissue & Cells DNA Isolation Kit to extract DNA from insects?

A. Yes, for tough samples like insects use the optional Proteinase K digest that comes with the kit and homogenize at high speed for three cycles of 20 seconds each. (3100 rpm on the PowerLyzer™ and 6100 rpm on a Fastprep or Precellys.)

Q.I would like to extract microbial DNA from infected tissue. What kit should I use?

A. Even though the UltraClean Tissue & Cells DNA Isolation Kit was originally designed for somatic cells, it can be used to isolate microbial cells as well. It has a bead beating step with garnet bead tubes, 0.7 mm, which will lyse open gram +/- bacteria as well as fungus. In order to minimize the amount of isolated tissue gDNA, leave out the proteinase K digest.

Arrow FAW DNA RNA Isolation questionsSwabs

Q. I am trying to extract microbial DNA from samples with PCR inhibitors collected on cotton swabs. What do you recommend?

A. Some swab samples will contain PCR inhibitors. Examples are fecal containing rectal swabs or dust containing swabs taken from environmental surfaces. To extract bacterial DNA from these swabs we suggest that you use the BiOstic Bacteremia DNA Isolation Kit. The BiOstic Bacteremia Kit employs our IRT method for removal of PCR inhibitors along with a strong lysis buffer. To use a swab with this kit start with the following protocol: 1. Add 450 ul of the CB1 buffer directly to the MicroBead tube. 2. Place and rotate the swab in the buffer and let it soak for a few minutes to release the cells into the solution. 3. If the swab has a head that can be snapped off, go ahead and do so, leaving the swab in the tube. Otherwise remove the swab now while gently squeezing it against the wall of the tube to remove as much of the solution as possible. 4. Proceed with the 70 C heating step and the rest of the normal protocol.

Q. I am trying to extract microbial DNA from relatively clean samples without PCR inhibitors collected on cotton swabs. What do you recommend?

A. For samples that do not have any PCR inhibitors you can use the UltraClean Tissue and Cells DNA Isolation Kit. It contains 2 ml bead tubes and one of our strongest lysis buffers for effective lysis of prokaryotic cells. To use a swab with this kit start with the following protocol: 1. Add 700 ul of Solution TD1 to the 2 ml Dry Bead Tube. 2. Place and rotate your swab in the buffer and let it soak for a few minutes to release the cells into the solution. 3. If the swab has a head that can be snapped off, go ahead and do so, leaving the swab in the tube. Otherwise remove the swab now while gently squeezing it against the wall of the tube to remove as much of the solution as possible. 2. Proceed with the vortex bead beating step and the rest of the normal protocol.

Q. I want to extract human/host DNA from samples with PCR inhibitors collected on cotton swabs. What do you recommend?

A. If your sample has a lot of PCR inhibitors and you want the human DNA (for example, a swab taken from a person who forgot to avoid eating or drinking before taking the sample), you can use the BiOstic Bacteremia DNA Isolation kit. The BiOstic Bacteremia Kit employs our IRT method for removal of PCR inhibitors along with a strong lysis buffer. To use a swab with this kit start with the following protocol: 1. Do not use the bead beating tube. Instead place and rotate the swab into a standard micro centrifuge tube containing 450 ul of Solution CB1 and let it soak for a few minutes to release the cells into the solution. 2. Remove the swab while gently squeezing it against the wall of the tube to remove as much of the solution as possible. 3. Vortex for 10 seconds to mix and place at 70 C for 15 minutes. Additionally, you can add 10 ul of proteinase K here to improve cell lysis. 4. Proceed with the rest of the protocol starting with the addition of 100 ul of Solution CB2 which removes the PCR inhibitory substances.

Q. I want to extract human/host DNA from relatively clean samples, without PCR inhibitors, collected on cotton swabs. What do you recommend?

A. If you are going for the eukaryotic/host DNA from a swabbed sample, and the sample doesn't have PCR inhibitors the Blood Spin DNA kit is a good choice. To use swabs with this kit start with the following protocol: 1. Place the swab in 400 ul of TE buffer (10 mM Tris, 1 mM EDTA) to cover your swab. 2. Rotate the swab in the buffer and let it soak for a few minutes to release the cells into the solution. 3. If the swab has a head that can be snapped off, go ahead and do so, leaving the swab in the tube. Otherwise remove the swab now while gently squeezing it against the wall of the tube to remove as much of the solution as possible. 4. Add 400 ul of Solution B1. Then add 10 ul of the proteinase K and let the sample digest for 30 minutes at 60C. 7. If the swab head is still in the tube remove it as described in Step 3. Add 800 ul of Solution B2 (100% ethanol) and then bind the DNA to the column in two spins adding 600 ul per spin. 8. Proceed with the rest of the protocol as directed.

Q. I want to extract RNA from contaminated buccal swabs or fecal swabs. What kit should I use?

A. The PowerMicrobiome RNA Isolation Kit is designed for fast and easy purification of total RNA from samples high in PCR inhibitors. Swabs should be frozen and stored at -80C before use to protect the integrity of the RNA. When ready to do the prep, place the frozen swab into bead tube containing 650 ul of Solution PM1/bME and allow to thaw. 2. Rotate the swab in the buffer and let it soak for a several minutes to release the cells into the solution. 3. If the swab has a head that can be snapped off, go ahead and do so, leaving the swab in the tube. Otherwise remove the swab now while gently squeezing it against the wall of the tube to remove as much of the solution as possible and proceed with the rest of the protocol.

Arrow FAW DNA RNA Isolation questionsBiofilm

Q. For what types of samples should I use the PowerBiofilm DNA Isolation Kit?

A. Biofilms can be found inside sink pipes, on lagoon rocks, on stream rocks, and as multi-layered sheets called microbial mats. Biofilms are composed of bacteria irreversibly attached to a substrate by extracellular polymeric substances or EPS. Within this matrix, a number of compounds can be found including humic substances, metals, salts, and pesticides. As a result, microbes within a biolm are difficult to lyse and the nucleic acids are often bound to numerous inhibitory substances. Because of this most commercial nucleic acid extraction kits are unsuccessful. The PowerBiofilm™ DNA/RNA Isolation Kits combine a biofilm pretreatment step with a powerful cell lysis solution and patented Inhibitor Removal Technology® to yield consistent, high quality, inhibitor free DNA and RNA. For an in depth description of DNA and RNA Isolation from biofilms see Application Note.

Q. What is the difference between the PowerBiofilm and PowerSoil® Kit?

A. The PowerBioFilm DNA Isolation Kit has a unique chemistry for melting the EPS in biofilm and has a bead tube with a bead mix for lysis of microbial mats. Both kits have IRT and use a silica column for purification. An in depth description of the differences can be found here.

Arrow FAW DNA RNA Isolation questionsFood

Q. If I want to extract DNA from fermented milk product and then continued for DGGE analysis, should I use the PowerFood or the PowerMicrobial DNA Isolation Kit?

A. We would recommend the PowerFood Microbial DNA Isolation Kit for fermented milk because it will be better at removing inhibitors in the milk.

Q. We want to extract fungal DNA from food without culturing (bread dough, milk, juice, dog food, beer, etc.) The sample contains a lot of inhibitors like starch, protein or fat. Can we use the PowerFood Microbial DNA Isolation Kit?

A. Yes for direct isolation of microbial DNA from solid or liquid food samples (high in inhibitors), you can use the PowerFood DNA Isolation kit (21000-50). Follow the protocol in the Hints and Troubleshooting Guide for isolating DNA directly from food without culturing

Arrow FAW DNA RNA Isolation questionsVirus

Q. We are interested in using the MO BIO PowerWater DNA Isolation Kit to isolate DNA/RNA from virus in water. However, the virus is too small to be captured by a 0.2 micron water filter. What kit should I use?

A. In order to extract DNA/RNA virus from a liquid, it must first be concentrated into a very small volume. We recommend volumes of 200 microliters or less so as not to dilute the lysis buffer. Options for concentrating the virus include using a specialized 0.02 micron aluminum oxide filter, a specially treated MgCl2 HA filter or positively charged and highly adsorptive media. Check out our blog article:Like Water for Virus.

Arrow FAW DNA RNA Isolation questionsPowerMag Kits

Q. I would like to use one of your PowerMag kits. However, I do not have an epMotion or a KingFisher robot. I have a different instrument. Can I still use your kit?

A. You might. We do not have program files for other robots but have simplified versions of our robotic commands in pdf format. We are happy to send these to you so that you can adapt them to your robot. Please contact technical support for assistance.

Q. I am using a PowerMag kit for the KingFisher robot. My robot uses software version 3.1 and expects protocol files with an ".msz" extension. However, the file on your website has extension ".bdz". Can I use this?

A. You will need to contact Fisher Scientific to get their latest software which accepts bdz files. There is no way to convert one type of file into the other.

Arrow FAW DNA RNA Isolation questionsGeneral

Q. When does my MO BIO product expire?

A. Each kit manufactured by MO BIO has the expiration date listed on the kit box label. If you have an individual component you can contact technical support with the lot number and we can tell you the expiration date. Using products after the expiration date can result in a reduction in performance.

Arrow FAW DNA RNA Isolation questionsShipping

Q. I extracted DNA using one of the MO BIO DNA Isolation kits. Now I need to ship my DNA to another location. Any tips on how to protect my DNA?

A. Microcentrifuge tubes and 96 well plates containing suspended DNA can easily break and contaminate other samples during shipping. To prevent this from occuring, special care must be taken. The Earth Microbiome Project has put together a set of standard procedures for shipping plates and microcentrifuge tubes that we found to be quite helpful. Read more

Arrow FAW DNA RNA Isolation questionsInstrumentation

Q. What is a Vortex adaptor?

A. It’s a detachable horizontal tube holder for efficient and consistent mixing of multiple samples for time periods too inconvenient to hold by hand. MO BIO Labs manufactures and sells adapters in a variety of tube sizes from 2 ml up to 50 ml.

Q. What is the MO BIO PowerLyzer™ 24?

A. The PowerLyzer™ 24 Bench Top Bead-Based Homogenizer is a high powered bead beating instrument designed for the most efficient and complete lysis and homogenization of any biological sample. In as little as 30 seconds, the PowerLyzer™ 24 homogenizer is capable of processing up to 24 samples in 2 ml tubes. Even the toughest and most difficult samples such as pine needles, seeds, spores, fungal mats, bone, and skin are easily and effectively lysed using the PowerLyzer™ 24 homogenizer and MO BIO's high-quality nucleic acid isolation kits.

Q. My new MO BIO Vortex Adaptor will not snap onto the Vortex. What's wrong?

A. If you haven’t used it before the Vortex Adaptor can be a little tricky to lock down. Many people are afraid they are going to break it and so don't push it down hard enough. Make sure to line up the flat edges of the connectors and that you hear a "snap" when you push the adaptor down. Click here to see a video demonstration of the MO BIO vortex adaptor.

Q. I am having difficulty getting the MO BIO vortex adaptor off of the Vortex. What's wrong?

A. If you haven’t used the Vortex Adaptor before, it can be a little tricky. However, pulling it off is easy if you use the right technique. Place your thumbs on the black button in the middle while wrapping your fingers around the edges and pulling up. Click here to see a video demonstration of the MO BIO vortex adaptor.

Q. Will your vortex adaptors fit my current vortex?

A. Maybe. Our vortex adaptors were designed to fit the MO BIO Vortex-Genie® 2. However, they are also compatible with other Vortex-Genie® 2 mixers, the Fisher vortex models: 02-215-365 through 02-215-376, and the VWR Brand vortex model: 58816-121. There may be others that work as well. Call or email Tech Support if you are unsure.

Q. I have a Precellys® 24. Can I use the MO BIO PowerSoil® DNA Isolation kits with a Precellys® 24?

A. Yes. Although, the PowerSoil® DNA Isolation kits have been optimized using the MO BIO PowerLyzer™ 24, they can also be used with other high powered bead beating instruments.

Q. I have a FastPrep® 24. Can I use the MO BIO PowerSoil® DNA Isolation kits with a FastPrep® 24?

A. Yes. Although, the PowerSoil® DNA Isolation kits have been optimized using the MO BIO PowerLyzer™ 24, they can also be used with other high powered bead beating instruments. 

Arrow FAW DNA RNA Isolation questionsDNA Clean-up

Q. What is the minimum size recovered using the UltraClean PCR Clean-up Kit?

A. 60bp

Q. What is the maximum fragment recovered using the UltraClean PCR Clean-up Kit?

A. Up to 10kb.

Q. Does the UltraClean PCR Clean-up Kit remove primer dimers?

A. Yes. Any DNA below 50 bp will be removed.

Q. I need to clean-up fragments larger than 10kb. Do you have something I can use?

A. Yes. For recovery of fragments larger than 10kb, use the silica based UltraClean 15.

Q. What is removed from the PCR reaction using the UltraClean PCR Clean-up Kit?

A. salts, primers, dNTPs, radio-labels, enzymes

Q. How much DNA / PCR product can I bind with the UltraClean PCR Clean-up Kit?

A. The silica membrane has been tested to bind a maximum of 20ug of DNA.

Q. I have no or diminished yields after cleaning up my PCR sample with the UltraClean PCR Clean-up Kit? What's wrong?

A. There are a number of possibilities. Did you make sure that the SpinBind was vigorously shaken before use? Shake Spinbind before use to maximize recovery. Did you take into account the dilution factor when eluting into 50ul? Is the SpinClean wash buffer fresh? The SpinClean wash buffer is mainly ethanol and may have evaporated so that the product is eluting off the column prematurely. Try preps again with fresh SpinClean.

Q. My 260/230s are low (below 1.0). What's wrong?

A. Carryover of guanidine salt may be a problem. Increase the spin time and speed during the wash steps. Try 2-5 minutes.

Arrow FAW DNA RNA Isolation questionsBlood

Q. Can I use the BiOstic Blood Total RNA Isolation kit to isolate the RNA from buffy coats/White blood cell (WBC) pellets?

A. Yes, you can use the BiOstic Blood Total RNA Isolation Kit to isolate RNA from your buffycoat or WBC pellet. Start the protocol at step 5, White Blood Cell Lysis, by thawing and resuspending the pellet in 300 ul of Solution BR2 (containing bME). For more information on working with blood (it was written around Halloween hence the title) see the following blog entry: Blood Sucks: How to Tame it Guide for Vampires and Molecular Biologists

Q. Which kit will work best for isolating DNA from Mycoplasma?

A. Since Mycoplasma lack a cell wall, mechanical lysis should not be necessary. The UltraClean BloodSpin DNA Isolation Kit should do the trick.

Arrow FAW DNA RNA Isolation questionsDNA Quantification, Purity and Storage

Q. I have a low 260/230 ratio (<1.5) from my extracted DNA. What does it mean?

A. A common cause of high 230 nm absorbance after a DNA extraction is the carry over of guanidine HCl. These salts help bind DNA to the silica spin column. Try washing the column a second time with the wash buffer. Another cause of a low 260/230 ratio can be a high carry-over of inhibitors (like humic acid) from the sample. If the 340 nm reading is > 0.1 then this may be the problem. You may have trouble with downstream applications like PCR. The MO BIO PowerClean® DNA Clean-Up kit is very effective at the removal of inhibitors with minor loss of DNA.

Q. Does the elution buffer in your MO BIO kit contain EDTA?

A. Most of our elution buffers contain Tris only because not all of our customers want it. If you want to add EDTA for long term DNA storage you may add it for a final concentration of 1 mM EDTA. Keep in mind that EDTA can interfere with some enzymatic reactions in downstream applications. In DNA that is diluted by 1/10 in the final reaction volume should not interfere.

Q. I am trying to load my DNA/RNA samples into an electrophoresis gel but they keep floating out of the wells. What's happening?

A. After washing the DNA/RNA on a spin column, the DNA/RNA are usually spun for several minutes to dry the column. If the spin column is not dried thoroughly there will be ethanol carry-over that will cause the samples to be too light to sink in the well. Ethanol can be removed using standard DNA precipitation techniques.

Q. I extracted DNA from soil and my 340 nm absorbance reading is high (>0.1). What does it mean?

A. A high 340 nm reading after DNA extraction from a soil sample plus a low 260/230 (< 1.5) ratio usually indicates that the extraction carried over humic acids. You may have trouble with downstream applications like PCR. If PCR inhibition does occur, diluting the DNA sample by 1/10th can often overcome the inhibition. The MO BIO PowerClean® DNA Clean-Up kit is very effective at the removal of inhibitors with a minimal loss of DNA. See Application Note.

Arrow FAW DNA RNA Isolation questionsRNA

Q. Can the RNA kits be used to isolate small RNAs, such as microRNA and siRNA?

A. Yes, this can be accomplished with a simple protocol change. For the purification of small RNAs, such as microRNA and siRNA, following the step of binding RNA to the column, increase the amount of 100% Ethanol added to 2 volumes. You will need to transfer the lysate to a larger tube in order to accommodate the additional volume of 100% Ethanol. You will need to supply additional 100% ethanol for this step. This will increase the number of times you need to load the column with supernatant.