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I am a first year science undergraduate and I am just asking this as a more theoretical question rather than for carrying out a protocol, so I would appreciate if answers do not involve lots of complicated chemical names.
I was wondering how one would go about quantifying the amount of DNA in a given band on a gel electrophoresis. Some ideas I have had so far are:
- Cut out the DNA band and extract the DNA. Quantify using some known method such as measuring the absorbance at 260nm of the DNA in solution to find the concentration.
- When the DNA is stained with ethidium bromide, clearly there will generally be more stain uptaken in a band region if it has more DNA so it will fluoresce with greater intensity when illuminated with UV light. However I would have reservations to using this method because I would think that the amount of stain uptaken would depend also on the state of the DNA (i.e. if it is supercoiled or not), but then again I do not know how much of an impact this could have on how much stain is uptaken. Of course, if you know that all of your DNA is in the same state then there shouldn't be a problem with this method I think. Although here you would have to be aware that the concentration of ethidium bromide in a certain band depends on the concentration of nucleotides there, which depends both on the number of the DNA fragments and their size (which is known from the position of the band). Compare this with the final method…
- Finally, if one is making the DNA for example using dideoxy chain termination/Sanger sequencing in PCR, then you can use a radioactively labelled primer. The intensity of the image on the radioautograph would then directly tell you the number of molceules of that DNA fragment present in the band.
I was wondering which of these are viable options to use in the lab, and why/why not.
All three methods could be used to measure the amount of DNA. However in practice, method 2 (estimation by dye brightness) typically works best in a normal workflow. It really depends on what you plan to do as your downstream application.
Problems with method 1:
- Yield from gel purification methods is sometimes finicky and prone to loss.
- Absorbance of remaining agarose can influence the results. DNA cleanup is a must.
Problems with method 2:
- Quantification is difficult as you must estimate the band brightness against a known standard (add a known quantity of your ladder and use that for estimation).
- Supercoiling of plasmids causes multiple bands to appear, so the estimation must be performed multiple times and then added (now it's less of an estimation and more a guess)
Problems with method 3:
- Working with radioactivity adds a significant amount of safety protocols into a simple procedure
- All products from radiolabeling must also be handled as radioactive material
- Using an image has the same issue as method 2 where you're estimating instead of actually quantifying.
- You could get more quantitative results from a scintillation counter, but then the products become more difficult to use because of purification from the scintillation fluid (maybe this is possible but I never tried it).
- You're limited to procedures which would incorporate the radiolabeled nucleotides
- If you do something like dA-tailing, then you're not quantifying the amount of DNA, but the amount of copies of a transcript.
Quantification of density of DNA bands - (Oct/27/2003 )
[COLOR=blue]Could you please inform me about quantifying the DNA bands that I got from gel electrophoresis. I have a gel documentation system that I can take the pictures of the band under UV light. Now, I am trying to quantify the density of the bands. I have a software program for that on pc, but the manual is very confusing. If anybody knows and tells it in a simple way or if there is some information sites, I would be very glad to learn.
Especially, I can not understand the noise substraction part.
Biotechnology and Bioengineering
I know there are many such software even photoshop can do the job. Each software differs in terms of how to use it. I have been using one called Gelquant which is pretty easy to use. So there is no general instructions on how to quantitate your bands, just read thoroughly the help file provided by your software.
Thank you pcrman, I have a program and manual of it but it is very confusing? what is the simpler one that you are using?
Running agarose and polyacrylamide gels
One of the most widely used tools in molecular biology, electrophoresis provides a simple, low-cost way to separate nucleic acids based on size for quantification and purification. Get some tips on running your gels. From here you can also access a detailed PAGE troubleshooting guide.
Electrophoresis with agarose and polyacrylamide gels is one of the most widely used tools in molecular biology. Gels provide a simple, low-cost way to separate nucleic acids based on size for quantification and purification.
Agarose gels can be used to resolve large fragments of DNA. Polyacrylamide gels are used to separate shorter nucleic acids, generally in the range of 1&minus1000 base pairs, based on the concentration used (Figure 1). These gels can be run with or without a denaturant. Gels that are run without a denaturant are referred to as native gels. The DNA or RNA will migrate at different rates, depending on its secondary structure. Native gels allow the DNA or RNA to remain double stranded. Adding a denaturant to the gel, such as urea, will generally make all of the nucleic acids single stranded. Secondary structure will not form in denaturing gels and, therefore, only the length of the DNA will affect mobility.
Different concentrations of agarose and acrylamide are necessary to optimize resolution of nucleic acids with different lengths. Suggested concentrations are shown below in Table 1.
Table 1. Gel concentrations for size separation.
|Agarose Gels||Polyacrylamide Gels|
|% agarose||Size Range for Optimum Resoultion (bp)||% acrylamide||Size Range for Optimum Resoultion (bp)|
Acrylamide is a potent neurotoxin and, in its powdered form, can easily be aerosolized. Make sure to wear the appropriate personal protection, including gloves and a mask, when weighing out the material. Many companies sell acrylamide dissolved in water or pre-cast gels. These products are slightly more costly but reduce the risk of acrylamide inhalation.
Ethidium bromide is the most common DNA stain available it is also toxic if inhaled, decomposes when heated to produce toxic gases, and is suspected of causing genetic defects . Always wear gloves and avoid microwaving liquids containing ethidium bromide. Non-mutagenic fluorescent dyes available as an alternative include Bio-Safe&trade (Bio-Rad), SYBR-safe&trade (ThermoFisher), and GelRed Nucleic Acid Stain (Phenix Research Products). While more costly than ethidium bromide, these stains reduce the need for isolating and decontaminating gel electrophoresis stations. Note that ethidium bromide only intercalates into double-stranded DNA and, therefore, is not a good stain for single-stranded DNA analysis.
Tips for acrylamide gel electrophoresis
- Use fresh Ammonium Persulfate (APS). APS catalyzes the polymerization of acrylamide. Using old APS or APS stored above -20°C will result in slow or incomplete polymerization. Keep small, fresh aliquots in the freezer.
- Know how your tracking dye(s) will migrate.In agarose gels, Bromophenol Blue and Xylene Cyanol will migrate at approximately 3000 and 300 bp, respectively. These dyes will migrate at different rates in acrylamide gels depending on the gel density. Table 2 provides the approximate migration rate in terms of the relative size of single-stranded/denatured DNA.
- TAE or TBE? Agarose gels commonly use Tris-Acetic Acid-EDTA (TAE) or Tris-Boric Acid-EDTA (TBE) buffers. TAE buffer has the advantage that it can be made in 50X stock solutions. However, it buffers less efficiently and, in some cases, its use results in smeared bands. TBE buffered gels yield sharper bands, particularly when using small-sized DNA fragments, and can be run at higher voltages. However, the borate in TBE can inhibit some enzymes&mdashincluding T4 DNA ligase&mdashin DNA purified from these gels.
- What voltage to use? Agarose gels can be run at a large range of voltages&mdashfrom 0.25&ndash7 V/cm. High voltages save time but can result in overheating of the gel, even leading to melting of low percentage agarose gels. High voltages can also cause band smearing, particularly of fragments >10 kb. The sharpest bands can be obtained by running gels in TBE overnight at 0.25&ndash0.5 V/cm.
Table 2. Dye migration rates in acrylamide gels.
|Nondenaturing gels||Denaturing gels|
|% Acrylamide||Bromophenol Blue (nucleotides)||Xylene Cyanol (nucleotides)||Bromophenol Blue (nucleotides)||Xylene Cyanol (nucleotides)|
*Adapted from Sambrook J, Fritsch EF, Maniatis T. (1989) in: Molecular Cloning: A Laboratory Manual, Cold Spring harbor Laboratory.
Troubleshooting gel electrophoresis
- Blurry bands? Too much DNA or excess salt will create smeared bands and/or streaking in the gel. Loading the correct amount of DNA (usually a maximum of 100&minus250 ng/mm well width) and desalting samples with a spin column prior to loading will prevent this.
- Bands in the wrong place?Do not heat nucleic acids before running on a native gel, and do not exceed 20 V/cm (measured from anode to cathode, rather than entire gel length) or allow the gel to exceed 30°C. For the sharpest bands, run the gel slowly, at 5 V/cm.
- Loading buffer floats away? Rinsing wells with running buffer just before loading is essential failure to do so may prevent the loading mixture from sinking to the bottom of the well, resulting in an uneven band and delayed migration.
Adam Clore, PhD, Director of Synthetic Biology Technical Support & Development, IDT.
Published Jun 17, 2011
Revised/updated Sep 20, 2017
DECODED online newsletter
Custom DNA & RNA Oligos
Your DNA or RNA sequence, desalted, deprotected, up to 1 µmol in tubes or plates. Verified by mass spectrometry.
Include mixed bases, modifications. Request purified, duplexed, premixed (RxnReady ® Pools).
Materials and Methods
Human biological samples and DNA preparation
Handling of human biological samples was carried out according to the national legal framework (Law on Biomedicine Research [July 2007]). The samples used were collected after informed consent of the donors and immediately anonymized. Local scientific and ethics committees approved the procedures performed in this work (32120017 project code). The samples used were (A) 118 frozen tissues in optimal cutting temperature (OCT) reactive (Tissue-Tek., Cat. No 4583), (B) 68 FFPE tissues, (C) 119 frozen EDTA blood samples and (D) 26 saliva samples collected in Oragene ® system (DNA Genotek, Inc. Cat. No. OG-250). The collection of samples was performed according to international recommendations and manufacturer's instructions.
For DNA isolation from high sample volumes, the paramagnetic beads based instrument Chemagic MSMI (PerkinElmer, Inc.) was used for each biospecimen. In brief, Chemagic DNA Blood Kit special (PerkinElmer, Inc. Cat. No. CMG-703-1) was used for tissue sections but with Proteinase K for tissue (PerkinElmer, Inc. Cat. No. 834) and Lysis Buffer 1 for tissue (PerkinElmer, Inc. Cat. No. 805). DNA samples obtained from FFPE tissues were additionally cleaned with QIAamp DNA Mini Kit (Qiagen Cat. No. 51304). Between 10 and 18 twenty micrometer sections for frozen tissues OCT and between 7 and 10 ten micrometer sections for FFPE tissues were used (the exact number of sections varied with the area occupied by the tissue after hematoxylin staining). Chemagic DNA Blood Kit special (PerkinElmer, Inc. Cat. No. CMG-703-1) was used for 5 mL of blood whose plasma fraction was replaced by PBS buffer. Finally, Chemagic DNA Saliva Kit special (PerkinElmer, Inc. Cat. No. CMG-1035) was used for 2 mL of saliva collected in Oragene system (DNA Genotek, Inc. Cat. No. OG-250). The corresponding Tris-HCl elution buffers available in the kits were used.
Quantification and DNA purity determination by spectrophotometry
Absorbance at 260, 280, and 230 nm for 2 μL of each DNA sample was measured in duplicate using the Nanoquant plate on the Infinite F200 instrument (Tecan Trading AG). The corresponding elution buffer was used as the blank. An additional measurement at 340 nm for each sample was automatically made by the instrument to bypass the absorbance values due to Nanoquant plate contaminants. The machine was calibrated and cleaned according to the recommended manufacturer's instructions.
Concentration of DNA from 260 nm absorbance was calculated by the instrument according to the Lambert–Beer law. The 260/280 ratio was used as the purity indicator of the DNA samples. Since an optimum value for 260/280 ratio for pure DNA is 1.8, the percentage of samples for each group with a purity ratio between 1.6 and 2.0 (1.8 ± 0.2) was additionally determined. The purity from the 260/230 ratio was also estimated for each DNA sample and it was represented versus DNA concentration.
DNA integrity analysis by electrophoresis
To observe DNA integrity, 50 ng of each DNA sample based on spectrophotometric measurement was analyzed by electrophoresis on a 0.8% agarose gel stained with GelRed (Biotium Cat. No. 41003). The Lambda-pUC Mix Marker 4 (Fermentas Life Sciences Cat. No. SM0291) was also separated as a size reference. Densitometry analysis was performed by setting a square area for the HMW band higher than 20 kb and the smear. The ratio between the densities of HMW band and smear areas was calculated for each DNA lane. The percentage of samples for each group with the presence of a HMW band was additionally determined.
Quantification and integrity estimation of DNA samples by PicoGreen
Quant-iT™ PicoGreen dsDNA Assay Kit (Life Technologies Cat. No. P7589) was used to quantify DNA by fluorescence. Lambda DNA contained in the kit was used to create a six-point standard curve from 3.125 to 100 ng/mL. DNA samples with a concentration determined by 260 nm absorbance higher than 100 ng/mL were diluted and later corrected through the dilution factor. Two microliters of each DNA and standard curve dilution were aliquoted into a CORNING 96 Flat Black plate (Corning, Inc. Cat. No. 3650). 1× Tris-EDTA (TE) buffer was used as negative control. PicoGreen reagent was diluted 1:200 in 1× TE buffer and 198 μL was added to each well. Samples were mixed and incubated 15 minutes in darkness before their fluorescence was measured with the Infinite F200 instrument (Tecan Trading AG).
To estimate DNA integrity, the ratio between extraction yields in micrograms calculated using PicoGreen and spectrophotometry was determined. For tissue sections, yield was normalized for different samples using the nuclear area in square millimeters examined through hematoxylin staining. In brief, nuclei were counted in the microscope and the area occupied in square millimeters was estimated using a graticule. The total micrograms of DNA obtained for each sample was divided by the estimated area.
Real-time PCR assay
For the real-time polymerase chain reaction (PCR) assay, 50 ng of DNA from each sample was amplified for the GAPDH and RPLP0 genes (PCR products of 87 and 69 base pairs [bp], respectively) in a LightCycler ® 96 System (Hoffmann-La Roche Ltd.) using the FastStart Essential DNA Green Master kit (Hoffmann-La Roche Ltd. Cat. No. 064027121001). A negative control was included in each assay. For each sample, duplicate determinations were made and the CT value was analyzed. The primer sequences for real-time PCR are shown in Supplementary Table S1 (Supplementary Data are available online at www.liebertpub.com/bio).
For PCR analysis, 50 ng of DNA from each sample was amplified for the ACVR2B, ZFX, AF4, and GAPDH genes. Twenty-five microliters amplification reactions contained 1 U Taq DNA polymerase (Qiagen Cat. No. 201205) dNTPs 0.15 μM (Thermo Scientific Cat. No. R0151, R0161, R0141, and R0171) for GAPDH and AF4, and 0.2 μM for ZFX and ACVR2B 0.4 μM of specific primers for GAPDH and AF4, and 0.8 μM for ZFX and ACVR2B Cl2Mg 1.5 mM (Qiagen Cat. No. 201205) for GAPDH, and 2.5 mM for AF4, ZFX, and ACVR2B and 0.5 ng/μL BSA (Sigma-Aldrich Co. Cat. No. B2518-10 MG), only for the ACVR2B reaction. PCR products of 5049, 1137, 400, and 87 bp, respectively, were analyzed by agarose gel electrophoresis. The GeneRuler™ 100 bp Plus DNA Ladder and GeneRuler 1 kb Plus DNA Ladder (Thermo Scientific Cat. No. SM0321 and SM1331, respectively) were also separated as size references. The primer sequences for PCR are shown in Supplementary Table S1.
DNA Ladders for Quantification & Size Determination
BioCat is pleased to offer an extensive range of DNA markers with defined ng amounts for each band. Our partners supplying these markers are Norgen and BioVision.
Quantitative DNA Ladders
• Accurate size determination
• Easy estimation of nanogram quantities
• Well-spaced bands with higher-intensity references
• Ready-to-use with no preparation
• Stable at room temperature for over 2 years
• 17 products from 25 bp – 24,000 bp
• 100 loads per tube
• NEW 500 loads pack size also available
Norgen’s Quantitative DNA Ladders have been genetically engineered so that the bands are of precise and discrete sizes. Well-spaced bands and higher-intensity reference bands facilitate visual analysis of the gel. These ladders are ideal for quantification due to the precise known ng amounts of each band.
See product brochure (link below) for an overview of Norgen´s markers and to check ng quantities per band.
For accurate mass determination of your DNA bands see attached guideline (link below).
The features of the different Norgen DNA Ladders are as follows:
MiniSizer 50bp DNA Ladder 25bp - 650bp For PCR product size and amount confirmation
PCRRanger 100bp DNA Ladder 50bp - 1000bp For assessment of a range of PCR product sizes
PCRSizer 100bp DNA Ladder 100bp - 1000bp For assessment of a range of PCR product sizes (Free sample available, use link below to request free sample)
FastRunner DNA Ladder 50bp - 2000bp Quick sizing of PCR products and restriction digests
LowRanger 100bp DNA Ladder 100bp - 2000bp Convenient clone identification
CloneSizer 100bp DNA Ladder 100bp - 2686bp For fast running times and accurate visual evaluation
MidRanger 1kb DNA Ladder 300bp - 5000bp Sizing larger PCR products and most cloning applications.
FullRanger 100bp DNA Ladder 100bp - 5000bp Good range for small and large cloning applications.
HighRanger 1kb DNA Ladder 300bp - 10000bp Size determination of digested DNA. (Free sample available, use link below to request free sample)
HighRanger Plus 100bp DNA Ladder 100bp - 10000bp Analyzing DNA over a wide range of sizes.
UltraRanger 1kb DNA Ladder 300bp - 24000bp Size determination of digested DNA with high molecular weight
More 100bp and 1 kb ladders are offered from our partner BioVision. A ready to use version with Image Green is also available from them. Moreover BioVision offers Safe Image DNA Stains a new and safe nucleic acid stain for the visualization of double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and RNA in agarose and polyacrylamide gels (see link below). Related Links
Protocol: Gel Purification
Follow the Agarose Gel Electrophoresis Protocol with the following amendments:
Note: Gel purification is most efficient with lower % agarose gels, so you will want to stay in the 0.7-0.8% range if possible.
Note: You will want nice crisp bands. This can be achieved by using a wider gel comb and running the gel at a lower voltage.
Note: You will want to have enough space around each band to cut without having DNA in other lanes contaminating your sample. To accomplish this, it is best to skip lanes between samples and between the ladder and nearest sample.
Note: To minimize the risk of DNA damage, it is best to limit the UV exposure of the DNA. Therefore, it is a bad idea to use a gel imager to take a picture of the gel before cutting out the bands and you will want to use long-wavelength UV for as short a time as possible to get the bands cut out.
Once you have run your gel, move it to an open UV box (be sure to wear proper UV protection - especially for your eyes!), remove it from any gel tray as plastic will block much of the UV and with a clean, sterile razor blade, slice the desired DNA fragment from the gel.
Note: To protect the UV box, it is a good idea to place the gel on a glass plate if available. Unlike the plastic tray, this will not significantly reduce the UV, but will protect the UV box from being cut by the razor blade.
Note: Try to get as little excess gel around the band as possible. To do so, it is often important to take the excised band, lay it down on the UV box and trim the top, bottom and sides with the razor blade. This is especially important during the DNA purification step, as many kits cannot handle more than a certain total volume of gel per reaction.
Place the gel in a labeled microfuge tube.
Using a scale, weigh the tube with the gel fragment after zeroing the scale with an empty tube. Alternatively, you can just subtract the weight of the empty tube from the weight of the tube with the gel fragment. The weight of the gel is directly proportional to its liquid volume and this is used to determine how much of each buffer to add during the DNA isolation step.
Finally, you will want to isolate the DNA from the gel. This is most commonly done with a commercial gel purification kit, such as the QIAquick Gel Extraction Kit. Always follow the manufacturer's instructions.
Note: It is usually important to determine the concentration of the DNA that you isolated before proceeding to your next intended step with the now gel purified DNA. Find more information about DNA quantification here.
The broad steps involved in a common DNA gel electrophoresis protocol:
1. Preparing the samples for running
2. An agarose TAE gel solution is prepared
TAE buffer provides a source of ions for setting up the electric field during electrophoresis. The weight-to-volume concentration of agarose in TAE buffer is used to prepare the solution. For example, if a 1% agarose gel is required, 1g of agarose is added to 100mL of TAE. The agarose percentage used is determined by how big or small the DNA is expected to be. If one is looking at separating a pool of smaller size DNA bands (<500bp), a higher percentage agarose gel (>1%) is prepared. The higher percentage of agarose creates a denser sieve to increase the separation of small DNA length differences. The agarose-TAE solution is heated to dissolve the agarose.
3. Casting the gel
The agarose TAE solution is poured into a casting tray that, once the gel solution has cooled down and solidified, creates a gel slab with a row of wells at the top.
4. Setting up the electrophoresis chamber
The solid gel is placed into a chamber filled with TAE buffer. The gel is positioned so that the chamber wells are closest to the negative electrode of the chamber.
5. Loading the gel
The gel chamber wells are loaded with the DNA samples and usually, a DNA ladder is also loaded as reference for sizes.
The negative and positive leads are connected to the chamber and to a power supply where the voltage is set. Turning on the power supply sets up the electric field and the negatively charged DNA samples will start to migrate through the gel and away from the negative electrode towards the positive.
7. Stopping electrophoresis and visualizing the DNA
Once the blue dye in the DNA samples has migrated through the gel far enough, the power supply is turned off and the gel is removed and placed into an ethidium bromide solution. Ethidium bromide intercalates between DNA and is visible in UV light. Sometimes ethidium bromide is added directly to the agarose gel solution in step 2. The ethidium bromide stained gel is then exposed to UV light and a picture is taken. DNA bands are visualized in from each lane corresponding to a chamber well. The DNA ladder that was loaded is also visualized and the length of the DNA bands can be estimated. An example is given in the figure below.
Genomic DNA Isolation
Yield, purity and integrity are essential to performance in downstream applications such as PCR and sequencing. Optimization of extraction methodologies is key for success with challenging sample types and demanding downstream applications. The purified target DNA should be free of contaminants, including proteins, other cellular components and undesired nucleic acids.
Specialized, sample-type specific purification kits may be needed for more complex and challenging samples that contain degraded DNA or a have low concentrations of DNA. Challenging sample types include FFPE tissue, plasma or serum containing cell-free DNA, forensic samples or any source where the sample quantity is limiting.
Promega was one of the first companies to provide kits for the purification of DNA, as well as plasmids, with over 30 years of experience in nucleic acid extraction. We offer a wide range of genomic DNA extraction kits suitable for a variety of sample types and throughput needs, producing high yields and high-quality DNA for use in your downstream applications. Our products cover a variety of throughput options and processing methods suitable to your specific needs&mdashfrom manual single-preps to small benchtop or large-scale automated systems.
Utilizing spin, vacuum or magnetic-based methods, our manual single-prep solutions are best for processing less than 24 samples at a time. If you are looking for an automated solution, our cartridge-based kits for use with Maxwell® Instruments can process up to 48 samples in the same run. We also offer fully automated high-throughput extraction options utilizing plate-based processing methods, fully compatible with liquid handling platforms.
Although techniques like Southern blotting, which require microgram amounts of DNA, are still performed in molecular biology laboratories, most assessments of chromosomal DNA is done by PCR-based technologies. These include monoplex or multiplex PCR, SNP arrays, analysis and real-time PCR, ddPCR and next-generation sequencing (NGS). These latter techniques use nanogram amounts of DNA per reaction. Regardless of the system chosen, Promega genomic DNA purification kits provide the required yields of high-quality DNA with minimal contaminants.
Manual Purification Systems
Promega offers genomic DNA isolation systems based on sample lysis by detergents and purification by various methods. These include both membrane-based systems (e.g., the single-column Wizard® SV Genomic DNA Purification System (Cat.# A2360, A2361) or the high-throughput, 96-well Wizard® SV 96 Genomic DNA Purification System (Cat.# A2370, A2371) and easily automated paramagnetic silica systems. All of these systems purify genomic DNA that is amenable for use in many downstream applications.
The Wizard® Genomic DNA Purification Kit (Cat.# A1120, A1125, A1620) is both a versatile and scalable system for isolating genomic DNA using a precipitation-based method. With this system alone, chromosomal DNA can be isolated from whole blood (5), plant leaf (6), Gram-positive (7) and Gram-negative bacteria (8), mouse tail (9) and yeast (10). Additional sample types like fungus (11), infected frog tissues embedded in paraffin (12), saliva (13) and flour beetles (14) have also been used successfully.
Not only is this genomic purification system successful with many sample types, it is also easily scaled for the quantity of starting material by adjusting reagent volumes to accommodate your needs.
Traditional Column-Based Systems
For single-column isolation, the Wizard® SV Genomic DNA Purification System provides a fast, simple technique for the preparation of purified and intact DNA from mouse tails, tissues and cultured cells in as little as 20 minutes, depending on the number of samples processed (up to 24 by centrifugation, depending on the rotor size, or up to 20 by vacuum). A vacuum manifold or a microcentrifuge is used for sample processing. With some modifications, whole blood can also be used with this isolation system (15). This is a silica membrane-based system, meaning there are limitations to the amount of material that can be loaded onto a single SV column up to 20mg of tissue (mouse tail or animal tissue) or between 1 × 10 4 and 5 × 10 6 tissue culture cells can be processed per purification. With more sample, the prepared lysate may need to be split among two or more columns to avoid clogging.
Figure 2. Amplification of genomic DNA isolated from various tissue sources using the Wizard® SV Genomic DNA Purification System. One microliter of purified genomic DNA was amplified using PCR Master Mix (Cat.# M7502) and mouse-specific IL-1&beta primers (1.2kb product). Reactions with Mouse Genomic DNA (Cat.# G3091 +C) and without DNA (&ndashC) were performed as positive and negative controls, respectively. Thermal cycling conditions were: one cycle of 3 minutes at 95°C followed by 30 cycles of: 95°C for 30 seconds, 60°C for 1 minute, 70°C for 1 minute and 30 seconds final extension at 70°C for 7 minutes 4°C soak. All lanes contained 10µl of reaction product separated on a 1% agarose gel. PCR products were visualized by ethidium bromide staining. &ldquoSpin&rdquo and &ldquoVacuum&rdquo designations indicate the protocol used for genomic DNA isolation.
The genomic DNA isolated with the Wizard® SV Genomic DNA Purification System is of high quality and performs well in agarose gel analysis, restriction enzyme digestions and PCR analysis as seen in Figure 2. Table 1 provides typical yields of genomic DNA purified from a variety of sources.
Table 1. Typical Genomic DNA Yield From Various Tissues using the Wizard® SV Genomic DNA Purification System.
|CHO cells||1 × 10 6||5µg|
|NIH/3T3 cells||1 × 10 6||9µg|
|293 cells||1 × 10 6||8µg|
Researchers have used this simple and rapid system for many additional sample types and applications including mosquitoes (16), mammary stem cells (17), Bacillus subtilis (18), Escherichia coli (19), the larval form of the Schistosoma mansoni parasite (20) and viral DNA from Kaposi&rsquos sarcoma herpes virus-infected BC3 cells (21).
For high-throughput, 96-well isolation, the Wizard® SV 96 Genomic DNA Purification System is available. Amplifiable genomic DNA can be isolated from up to 5 × 10 6 cells, 20mg of tissue or up to 1.2cm of a mouse tail tip without centrifugation of the lysate prior to purification.
This multiwell system requires a vacuum manifold (Vac-Man® 96 Vacuum Manifold, Cat.# A2291) and a vacuum pump capable of generating 15&ndash20 inches of mercury or the equivalent. Genomic DNA was isolated from three different source types then used in a monoplex PCR and run on an agarose gel as shown in Figure 3. Figure 4 compares the yield from the three Wizard® SV Genomic DNA purification methods (96-well plate, vacuum and centrifugation).
Figure 3. Agarose gel electrophoresis of PCR products amplified from 1µl of mouse tail, CHO cells and tomato leaf sample genomic DNA isolated using the Wizard® SV 96 Genomic DNA Purification System. A total of 10µl of PCR product is visualized on a 1.5% agarose gel stained with ethidium bromide. Panel A. IL-1&beta (1.2kb) amplified from mouse tail. Panel B. &beta-actin (250bp) amplified from CHO cells. Panel C. Chloroplast DNA (600bp) amplified from tomato leaf. Lane M, 1kb DNA Ladder (Cat.# G5711).
Figure 4. Comparison of DNA yields using the Wizard® SV and SV 96 Genomic DNA Purification Systems. Average yield of genomic DNA in micrograms purified from 20mg mouse tail clippings. The average A260/A280 ratios are: SV 96, 1.7 ± 0.08 SV vacuum method, 1.7 ± 0.14 SV spin method, 1.7 ± 0.14.
High-Performance Column-Based Systems
We offer two different ReliaPrep&trade gDNA Miniprep Systems that purify genomic DNA using a cellulose column-based method: ReliaPrep&trade Blood gDNA Miniprep System (Cat.# A5081, A5082) and ReliaPrep&trade gDNA Tissue Miniprep System (Cat.# A2051, A2052). Both are ready-to-use systems that obtain intact genomic DNA without using ethanol washes or precipitations. The ReliaPrep&trade Blood gDNA Miniprep System processes 200&mul of blood or body fluid, either fresh or frozen, in less than 40 minutes. Yields from blood are typically 4&ndash10&mug, depending on the white blood cell count. Up to 25mg of tissue, a buccal (cheek) swab or a 1cm mouse tail can be processed with the ReliaPrep&trade gDNA Tissue Miniprep System and the eluted DNA recovered in 30 minutes or less. The purified DNA can be eluted in as little as 50µl and is suitable for use in downstream applications such as RT-qPCR.
Figure 5. The yield of genomic DNA from the ReliaPrep&trade Blood gDNA Miniprep System varies with white blood cell count. Whole blood was obtained from several individuals, and white cell counts were determined using a hemocytometer. Two hundred microliters of blood was used for genomic DNA purification (n = 3 or 4), and the amount of isolated gDNA was quantitated by absorbance spectroscopy.
Figure 6. Comparison of elution volume with concentration, yield and purity. Aliquots of blood (200&mul) were processed using the ReliaPrep&trade Blood gDNA Miniprep System (n = 4) and eluted with 30&ndash200&mul of Nuclease-Free Water. Concentration (Panel A), total yield (Panel B) and purity (Panel C) were assessed using absorbance spectroscopy. Yield decreased slightly with decreases in elution volume, while concentration increased. Purity as measured by optical density ratios remained constant.
Automated Systems for DNA Purification
As laboratories try to improve productivity for research, diagnostics and applied testing, the need has increased for easy-to-use, low- to moderate-throughput automation of purification processes. Automation eliminates the hands-on time and labor of manual purification, giving you more time and energy to focus on your research.
Traditionally, automation refers to the use of large, specialized and costly equipment that requires extensive training to operate and maintain. Promega has developed the Maxwell® Systems, which provide flexible, reliable, compact and easy-to-use alternatives to traditional automated systems.
The Maxwell® Systems are designed for efficient, automated purification from a wide range of sample types (see Table 2). Maxwell® Instruments are supplied with preprogrammed automated purification methods, and can process up to 48 samples in as little as 30–40 minutes (depending on instrument, sample type and method). The purified concentrated DNA or RNA are high quality and high yield, making them compatible with many common downstream applications, including qPCR, ddPCR, genotyping, sequencing and NGS.
Figure 7. The Maxwell® RSC (left) and Maxwell® RSC 48 (right).
Table 2. DNA yield from various sample types after purification using the Maxwell® RSC Instrument and DNA Purification Kits.
Up to 50mg of liver tissue
Up to 50mg of lung tissue
Maxwell® Kits offer predispensed reagent cartridges for purification of genomic DNA, RNA and Total Nucleic Acid. Application and sample type-focused kits make the Maxwell® Instruments a versatile extraction instrument for laboratories that may work with one or all of these different applications.
Figure 8. The Maxwell® RSC DNA or RNA extraction methods start with cartridges prefilled with purification reagents and paramagnetic particles, ready for your samples. After sample addition, the Maxwell® RSC moves the paramagnetic particles and associated nucleic acids through multiple steps ultimately yielding highly pure RNA or DNA in 30–100µl.
The Maxwell® Systems purify samples using paramagnetic particles (PMPs), which provide a mobile solid phase that optimizes sample capture, washing and elution of the nucleic acid. The Maxwell® Instruments are magnetic-particle-handling instruments that efficiently bind nucleic acids to the paramagnetic particle in the first well of a prefilled cartridge. The samples are processed through a series of washes before the nucleic acid is eluted. The systematic magnetic particle-based methodology used by the Maxwell® Instruments avoid common problems associated with automated liquid handler-based purification systems, such as clogged tips or partial reagent transfers, which can result in suboptimal purification processing.
The benchtop-compact Maxwell® Instruments are easy to set up and require no special training for use. Optimized automated methods are preloaded, the prefilled reagent cartridges are snapped into place, your sample is added and you select "Start" to begin the appropriate method. A full list of nucleic acid extraction kits is available here.
Several Maxwell® Instrument reagent kits are available and allow optimal extraction from a variety of sample types, including blood, serum and plasma, formalin-fixed, paraffin-embedded (FFPE) tissue, bacteria, plant, food and animal tissue.
Maxwell® HT Systems allow purification of DNA or RNA at scale on any laboratory liquid handler in 24- or 96-well SLAS format. Maxwell® purification chemistries use novel magnetic particle-based solutions that naturally decrease contamination carryover.
In addition to trusted chemistry, you&rsquoll gain expert support to get started with automation or optimize your current HT workflow. Our team of automation experts can offer assistance with most of the leading laboratory automation providers in the world and help you develop and implement an automated nucleic acid purification solution customized to the needs of your laboratory.
Genomic DNA Extraction Kits
Looking for extraction options by sample scale or type? Explore our DNA extraction portfolio to discover the right solution for your purification needs.
Scalable Automation Solutions
The Maxwell® RSC Instruments provide a compact, automated nucleic acid purification platform that processes up to 16 (Maxwell ® RSC) or up to 48 (Maxwell ® RSC 48) samples simultaneously.
High-throughput Purification Chemistries and Automation Support
Maxwell® HT chemistries allow automation of nucleic acid purification on liquid handlers. Our team of automation experts offer assistance to help develop and implement an automated nucleic acid purification solution customized to the needs of your laboratory.
High-Throughput Systems for Genomic DNA Isolation
Promega offers several automated high-throughput options to isolate genomic DNA isolation from blood samples. Some laboratories, such as biobanks, have a desire to isolate DNA from large amounts of starting material (e.g., 10ml of blood). The ReliaPrep &trade Large Volume HT gDNA Isolation System (Cat.# A2751) provides an effective means for isolation of genomic DNA derived from blood fractions derived from 2.5&ndash10ml samples of whole blood. This chemistry can be automated onto liquid handlers by using a Promega HSM device, which enable processing of purification reactions in 50ml conical tubes.
Liquid level sensing and instrument operating software scale the chemistry to sample input volume for each individual sample, reducing reagent waste and expense. The automated system can also process sample in 14ml tubes using the Low Volume Adapter XAT1020 (LVA and Methods) which enables processing samples from 0.25&ndash3ml.
There are no tedious centrifugation steps or hazardous chemicals, which are inherently handling workstation, offering walkaway purification of genomic DNA from whole blood, regardless of sample storage or shipping conditions.
Figure 9. DNA was isolated from whole blood via three methods, separated by CHEF gel electrophoresis and visualized by ethidium bromide staining. DNA isolated using the ReliaPrep&trade Large Volume HT gDNA Isolation System provided DNA with a size range of 20–125kb precipitation-based purification isolated DNA with a size range of 20–200kb while column-based methods demonstrated gDNA with a size of 20–75kb.
There is an option for low-throughput isolation of gDNA from up to 32 samples at one time when the Heater Shaker Magnet Instrument (HSM 2.0 Cat.# A2715) is used on a bench versus integrated on a liquid handler where the user dispenses and aspirates reagents from the samples as directed by the software on a computer screen. The preprogrammed methods control the heating, shaking, magnetization and timing of the steps required for the semi-automated purification.
In addition to whole blood, a variety of other sample types can also be processed, including stabilized saliva, buccal wash samples, blood fractions, buffy coats, red cell pellets and all cell pellets. For fully automated purification, the HSM 2.0 Instrument can be integrated with a robotic liquid-handling workstation.
Automating reagents onto instrumentation requires a carefully planned and executed approach. Collaborating with Promega gives you access to scientists who have designed automated purification for hundreds of labs, across a wide range of sample types.
Automating reagents onto instrumentation requires a carefully planned and executed approach. Collaborating with Promega gives you access to scientists who have designed automated purification for hundreds of labs, across a wide range of sample types.
Figure 10. Automated DNA yields for blood fractions. DNA yield is linear with respect to original volumes of blood. Panel A. DNA yields as determined by NanoDrop spectrophotometer. Panel B. DNA yields as determined using the QuantiFluor&trade dsDNA System. All samples were prepared from a single donor. Manual samples were processed using the Wizard® Genomic DNA Purification Kit. Each point is the mean of n=4 values with error bars of 1 standard deviation.
Custom HT Nucleic Acid Purification
Implementing automated nucleic acid purification technologies onto your high-throughput workflow can be challenging and time-consuming. Our Field Support Scientists can provide the support you need to get started.
Selected DNA Purification Kits by Sample Type
Learn more about some of our specialized kits below, and explore the breadth of our portfolio and compare our DNA extraction kits with the help of our product comparison page to discover the right solution for your DNA purification needs.
Fixed-Tissue Genomic DNA Isolation
The MagneSil® Genomic, Fixed-Tissue System (Cat.# MD1490), provides a fast, simple technique for the preparation of genomic DNA from formalin-fixed, paraffin-embedded tissue. After an overnight Proteinase K digestion, genomic DNA can be manually purified from FFPE thin tissue sections in less than an hour. Amplifiable genomic DNA can be isolated from 10μm sections without centrifugation of the lysate prior to purification. Up to 12 samples can be processed in the manual format using a MagneSphere® Technology Magnetic Separation Stand (Cat.# Z5332, Z5342).
Figure 11. Analysis of DNA purified from paraffin-embedded, formalin-fixed 10µm thin sections using the MagneSil® Genomic, Fixed Tissue System. Purified DNA was amplified, and the amplification products were analyzed on an ABI PRISM® 310 or 3100 genetic analyzer. Panel A. Amplification with a set of 16 fluorescently labeled primers. Amplification products range in size from 104 to 420 bases. Panel B. A 972-base fragment amplified using an amelogenin primer set. Panel C. A 1.8kb fragment amplified from the Adenomatosis polyposis coli (APC) gene. Increasing the extension time during amplification may help to balance yields between small and large amplification products and increase yields for large amplification products. Results will vary depending on the degree of cross-linking due to formalin fixation.
One advantage this system has over other purification methods, such as phenol:chloroform extraction, is its ability to remove most inhibitors of amplification, including very small fragments of DNA. Tissue that has been stored in formalin for extended periods of time may be too cross-linked or too degraded to perform well as a template for amplification. Figure 11 shows an amplification of 16 short tandem repeat (STR) loci and demonstrates how well the isolated DNA can work in multiplex PCR using the PowerPlex® 16 HS System (Cat.# DC2101, DC2100).
The Maxwell® RSC FFPE Plus DNA Kit (Cat.# AS1720) is an automated method for purifying up to 48 samples of one to ten 5μm sections of FFPE tissue samples on the Maxwell® RSC Instrument (Cat.# AS4500 1–16 cartridges per run) or Maxwell® RSC 48 Instrument (Cat.# AS8500 1–48 samples per run). The FFPE Plus chemistry is designed to provide high yield of DNA from FFPE when measured by spectroscopy that is suitable for amplification applications including qPCR, multiplex PCR and NGS. The protocol provides flexibility with either a 1-hour quick deparaffinization or 24-hour overnight protocol to fit your work flow needs.
The Maxwell® RSC DNA FFPE chemistry is Promega’s latest FFPE technology and has been designed to provide highly amplifiable DNA. Save time and labor by utilizing either FFPE chemistry with the Maxwell® Instruments, and avoid exposure to hazardous xylene utilized in other FFPE purification products. Our quality testing has also demonstrated virtually no PCR inhibitors in purified DNA samples, making your PCR and other downstream applications a breeze.
Utilizing the same chemistry as the Maxwell® RSC FFPE DNA, the Maxwell® HT DNA FFPE Isolation System (Cat.# A6372) provides a simple and reliable method for high-throughput, rapid isolation of genomic DNA from FFPE tissue samples. The system does not require an organic solvent, making it safe and convenient to use, and the purified DNA can be used directly in a variety of downstream applications, including PCR and NGS.
The Maxwell® HT DNA FFPE Isolation System purifies nucleic acid using paramagnetic particles, which provide a mobile solid phase to optimize binding, washing and purification of gDNA. The use of paramagnetic particles for DNA isolation eliminates the need for centrifugation or vacuum manifolds, making the system suitable for full automation.
As FFPE samples can have widely varying quality due to the nature of the sample fixation and embedding process, QC of samples can be an important part of the FFPE workflow.
Figure 12. Comparative data of the Maxwell® RSC DNA FFPE chemistry versus the Maxwell® RSC FFPE Plus DNA chemistry. The Maxwell® RSC FFPE Plus DNA method has been observed to produce more yield by absorbance and fluorescence, while the Maxwell® RSC DNA FFPE method produces more yield by PCR.
Spectrophotometry is a common way to evaluate the quality of extracted DNA and RNA. Most laboratories have a NanoDrop Microvolume Spectrophotometer (or similar device) and they are incredibly easy to use. Pipette 1-2µl of sample, select “Analyze” and the instrument provides a read out of concentration and purity via A260/A280 and A260/A230 ratios in just a few seconds. These devices have revolutionized routine sample quantitation in the lab, but is it the best method for assessing FFPE samples? There are two main considerations when using a NanoDrop: sensitivity and integrity. FFPE samples can have a wide-ranging yield of DNA or RNA often as little as 10ng or less in a volume ranging from 10µl to 100µl from an extraction. This can result in sample concentrations below the NanoDrop’s linear range. In addition, as a spectrophotometer, it does not differentiate between RNA, DNA or free nucleotides, which can result in dramatic inaccuracies in DNA/RNA concentration measurements. Finally, there is no way to determine if a sample is accessible to downstream enzymatic assays since it cannot detect the presence or absence of crosslinks (or other damage) within a sample.
Dye-Based Quantitation like the Promega QuantiFluor® dsDNA System (Cat.# E2670, E2671), provides a rapid and significantly more sensitive method to quantitate dsDNA or RNA compared to absorbance spectroscopy. This method provides a broadly useful estimate of concentration. When considering FFPE samples, it is important to note that dye-based quantitation does not estimate the integrity of the DNA/RNA or the extent of cross-linking in the sample, which could affect success in downstream assays.
Sizing Assays (e.g., agarose gel, Tape station, fragment analyzer, DV200) can provide an estimate of concentration and—more importantly—information on the size distribution of the fragments in the sample. FFPE-derived DNA, due to the fixation process, can be significantly fragmented compared to DNA from freshly frozen tissue. Below is a fragment analyzer trace (Figure 13) and associated DV200 scores (Table 3) of DNA isolated from FFPE sections using five different purification methods. While the sizing traces do assess the distribution of DNA size purified, it does not measure the degree of cross-linking within the sample or the presence of inhibitors.
Figure 13. Fragment analyzer trace of DNA isolated from FFPE sections using five different purification methods.
Table 3. DV200 scores of DNA isolated from FFPE sections using five different purification methods in fragment analyzer trace (Figure 13).
|Method||1 (Light Green)||2 (Blue)||3 (Red)||4 (Orange)||5 (Green)|
For example, when the same samples were quantitated by qPCR assays of various targets and fragment sizes, the yield by qPCR does not correlate well with the DV200 scores. In fact, in this example, the samples with the lowest DV200 scores had the greatest yield by qPCR (Figure 14).
Figure 14. qPCR yields of DNA isolated from FFPE sections. The same samples of DNA isolated by five different purification methods in the fragment analyzer trace and DV200 table above were quantitated by qPCR assays of various targets and fragment sizes.
While there are general trends, the DV200 score does not necessarily correlate with success in downstream assays such as qPCR.
qPCR has several advantages for the quantitation of FFPE samples. First, qPCR can be very sensitive, requiring only a small amount of sample and detecting pg/µl amounts of DNA. In terms of sensitivity in nucleic acid detection, it is surpassed only by ddPCR. qPCR can also provide a measure of how degraded or crosslinked a DNA sample may be since nucleic acid must be a suitable substrate for a DNA polymerase for a signal to be generated. Absorbance may not represent the sample suitable for the downstream assay because it will detect DNA, fragmented DNA and nucleotides. Finally, most qPCR QC assays, such as the ProNex® DNA QC Assay (Cat.# NG1004, NG1005) provide internal controls which are used to detect the presence of inhibitors in the sample prior to attempting a more expensive assay. This can help you assess not only the integrity of the nucleic acids, but also the likelihood of an amplification-based assay to be successful.
NGS is another assay used by some labs to QC their samples. There are several reasons for this. Some labs are trying to get as much data as possible from very precious samples, in which case any sequence information may be worth the expense and risk of failed sequencing runs. As a QC test, NGS may provide a lot of information, but it is expensive and can require large amounts of sample and time. Some labs run low pass NGS, which uses highly-multiplexed samples to lower the cost per sample to determine if it is worth the time and resources to sequence deeper. Most sequencing and purification providers recommend qPCR assays to quantitate FFPE DNA, as all NGS workflows depend primarily on the success of enzymatic amplification steps to obtain sequencing-ready DNA as part of library preparation steps.
Table 4. Comparative Pros and Cons of Various QC Assays.
|Method||Speed||Sensitivity||Quantitative?||Measure Purity?||Assess size of NA?||Detect Cross- linking?||Cost|
|Spectrophotometry (NanoDrop)||+++||+||Semi - quantitative||+||-||-||$|
|DV200||++||+||Semi - quantitative||-||+*||-||$$|
|Gel Electrophoresis||++||+/-||Semi - quantitative||-||+*||-||$|
Plant Genomic DNA Isolation
The Wizard® Magnetic 96 DNA Plant System (Cat.# FF3760, FF3761) is designed for manual or automated 96-well purification of DNA from plant leaf and seed tissue. The Wizard® Magnetic 96 DNA Plant System has been validated with corn and tomato leaf as well as with canola and sunflower seeds. The DNA purified from these samples can be used in PCR and other more demanding applications, such as RAPD analysis. Since plant materials can be particularly challenging to lyse, especially when working with tough or woody tissues, additional required equipment includes not only a magnet (MagnaBot® FLEX 96 Magnetic Separation Device, Cat.# VA1920) but also a device capable of breaking up seed or leaf material (e.g., Geno/Grinder® 2000 from SPEX CertiPrep, Inc.).
The yield depends on the source material and how well the seeds or leaf disks are pulverized prior to the genomic DNA isolation. Yield may range from 10–100ng from a single 8mm leaf punch. To increase the yield from the Wizard® Magnetic 96 DNA Plant System, a scale up in volume with up to 5 leaf punches can be used [as demonstrated in Promega Notes 79]. The potential scale-up is limited by the volume in a deep-well, 96-well plate.
Another automated option we have to meet your plant DNA extraction needs, is the Maxwell® RSC Plant DNA Kit (Cat.# AS1490). The Maxwell® RSC Plant DNA Kit is used with the Maxwell® RSC and RSC 48 Instruments to provide an easy method for efficient, automated purification of genomic DNA (gDNA) from a range of plant tissue samples, including corn, soybean and Arabidopsis. The Instruments are supplied with preprogrammed purification methods and uses predispensed reagent cartridges, maximizing simplicity and convenience.
Using this system, DNA can be purified from plant samples in under 60 minutes with minimal preprocessing and no organic extractions. Automated purification results in consistent purification, with less variability than traditional DNA extraction methods such as CTAB and spin-columns. The resulting purified DNA is ready to use in downstream applications, including amplification assays.
Serum-Plasma Genomic DNA Isolation
For high quality, purified cell-free DNA from plasma samples, we offer the Maxwell® RSC ccfDNA Plasma Kit (Cat.# AS1480). Utilizing the simple three-step protocol, the Maxwell® RSC Instrument can process 1 to 16 samples, and the Maxwell® RSC 48 Instrument can process 1 to 48 samples. Simply add 0.2–1.0ml of plasma to the prepared cartridges and select Start, no preprocessing of samples required. In approximately 70 minutes, you will have high yields of amplifiable DNA that is ready to be used in downstream assays including qPCR, NGS and digital PCR.
As a magnetic particle mover, not a liquid handler, the Maxwell® RSC additionally offers several advantages over other automated systems. Since no liquid handling or splashing occurs during sample processing, there is minimal risk of sample cross-contamination. It also eliminates the worry of potential clogs and inevitable system breakdowns that follow, ensuring a smooth workflow with fewer disruptions.
Bacterial Genomic DNA Isolation
If you need to make quick decisions about potential food contamination and spoilage, the Maxwell® RSC PureFood Pathogen Kit (Cat.# AS1660) offers a simple automated protocol with minimal hands-on steps. The kit effectively eliminates laborious sample preprocessing steps such as enzymatic pretreatment, as it works with inhibiting sample types and also has the ability to lyse both Gram+ or Gram– bacteria.
By coupling the high-performance Maxwell® chemistries with the trusted benchtop Maxwell® RSC instruments, you will be able to effectively purify bacterial DNA from up to 48 food samples in as little as 40 minutes. Once extracted, the resulting DNA is ready for advanced downstream molecular analyses, including serotyping, NGS and identification of spoilage organisms.
This method can be utilized for both raw and processed food and has successfully been used to isolate pathogen DNA from a wide variety of food samples, including E. coli 0157:H7 from uncooked beef, Salmonella enterica from uncooked chicken and Listeria monocytogenes from whole milk. Figure 15 below highlights a comparison of total DNA versus E. coli 0157:H7 DNA extracted from cilantro samples that were spiked with the E. coli 0157:H7 bacteria.
Figure 15. Comparison of total DNA and E. coli 0157:H7 DNA extracted from cilantro samples spiked with the indicated amounts of E. coli 0157:H7 bacteria. The total DNA concentration was assessed using the QuantiFluor® ONE dsDNA System.
Buffy Coat Genomic DNA Isolation
The Maxwell® RSC Buffy Coat DNA Kit (Cat.# AS1540) provides a simple, automated method of genomic DNA extraction using the convenient, prefilled cartridge format of the Maxwell® RSC Instrument. The kit contains all the reagents you need for optimal DNA extraction, and is compatible with blood stored in EDTA, heparin and citrate anticoagulants. Avoid the tedious and time-consuming hassle of preprocessing samples, simply add 50–250μl of your sample directly into well #1 of the cartridges. Your purified DNA is ready for analysis in about 50 minutes, and can be used directly in various downstream applications, such as agarose gel electrophoresis.
Food Genomic DNA Isolation
Food and plant materials often provide the greatest challenge for cell lysis and intact DNA extraction, due to the lysis conditions required to liberate the nucleic acid and the processing of plant materials into comestibles.
Another specialized genomic DNA isolation system is the Wizard® Magnetic DNA Purification System for Food (Cat.# FF3750, FF3751). This convenient protocol is designed for the manual purification of DNA from a variety of food samples including corn seeds, cornmeal, soybeans, soy flour and soy milk, generating results in one-third of the time of traditional methods. In addition, DNA can be purified from processed food such as corn chips, chocolate and chocolate-containing foods, lecithin and vegetable oils if used with the appropriate optimized protocols.
The DNA purified from many of these samples can be used in PCR-based testing for Genetically Modified Organism (GMO) DNA sequences, such as by quantitative analysis using TaqMan® assays. As with all isolation systems using the MagneSil® PMPs, a magnetic separation stand is needed and enables processing of up to 12 samples per batch. With samples containing highly processed food, the genomic DNA isolated will be fragmented and better suited for analysis using amplification rather than a Southern blot. The yield of DNA from this system will vary depending on source type and extent of food processing.
The Maxwell® RSC PureFood GMO and Authentication Kit (Cat.# AS1600) provides an easy and automated method for efficient purification of DNA for PCR-based food and ingredient authentication. The Kit is used with the Maxwell® RSC and RSC 48 Instruments and can purify DNA from raw and processed food samples, including corn, soybeans, canola, ground beef and ground pork.
100 minute protocol requires only 30 minutes of hands-on time, effectively achieving not only faster results with walk-away automation, but also freeing up laboratory resources for higher value activities. The purified DNA extracted using the PureFood Kit is ready to be used for several applications, including real-time PCR, gel electrophoresis, next-generation and Sanger sequencing and microarrays.
Nucleic Acid Purification Protocols for Plant & Food Sample Types
Explore our collection of protocols for manual and automated DNA or RNA extraction from a variety of food and plant samples.
Quantifying DNA in a band on gel electrophoresis - Biology
In this experiment we are given the scenario of a crime scene in a classroom where we will be analyzing DNA samples of evidence found in the classroom. After analyzing this data and comparing the data to the blood that was found on the ground in the classroom, we’ll be able to determine whose blood is it. The suspects are Mr. Gladson, Bobby, and Ms. Mason. The evidence found were a wad of bubble gum, a shard of glass with a reddish stain, a discarded tissue and a paper cup with lipstick stains. From the data we collected we’ll be able to analyze it via electrophoresis and find out if the blood on the ground belongs to either one of our suspects. Our hypothesis is that after we analyze the evidence one will match the blood on the floor and point to one of the suspects. As for our null hypothesis if none of them were to match the blood on the ground, they would be cleared and no longer remain suspects and that there was someone else in the room besides the three suspects.
We started by creating an agarose solution to create a gel for electrophoresis. After creating our solution we tape the edges of a tray so we can cast the solution in the tray and create our gel. The comb is to be put in the side that is closest to the negative cathode in the gel box. When the gel is casted the comb makes 8 wells that can be filled with our DNA samples.
We prepared our DNA samples by placing the sample DNA into 3 separate tubes and placed different restriction enzymes and marked each tube with the initial of each enzyme. We also had a control DNA as well as the DNA from the crime scene and added dye to each one and mixed it in a centrifuge for a couple of seconds. We then placed each sample of DNA, with the wells on the left side, in the first 5 wells. We used a micropipette to carefully fill each well with 15 microliters of each sample. The control sample from the crime scene went in well 1 and the next four are put in any order but make sure to keep track of which one is in which well. We then plugged in the gel box and set the box at 50 volts for 2 hours and what it does is it makes the dye move to the opposite side since opposite forces attract and DNA is negative it will move towards the positive anode. After two hours we took out our gel and moved it into a plastic tray and dyed it so the dye would attach to the DNA and left it overnight and let osmosis take place. The next day we drained out our containers and placed regular tap water into our trays and placed it back on the rocker for 15 minutes and we let reverse osmosis take place and then we took our gel and placed it on a light source tray to reveal the bands of DNA in the gel. We then took a ruler to measure how far each band traveled from each well and how many times it split during the process. Once were finished we matched up which sample matched the sample from the crime scene.