[{"data":1,"prerenderedAt":-1},["ShallowReactive",2],{"$fxLN3MUwXCdr5RPjwZYIDpOj8CHyjOmngWTgoKXPtZbg":3,"$fY3XZ65MF3Gf18MI2XDY7BtmLbFFMA5TFTVjqyynpppo":32,"$f3Ft0rKFJHppdzE-vuveecxx1BUcg9iOlMLtyzf_MJDg":238},[4,8,12,16,20,24,28],{"title":5,"slug":6,"path":7},"About Microbeonline.com","about-microbeonline-com","\u002Fabout-microbeonline-com\u002F",{"title":9,"slug":10,"path":11},"About Me","about-me","\u002Fabout-microbeonline-com\u002Fabout-me\u002F",{"title":13,"slug":14,"path":15},"Advertise with Us","advertise-us","\u002Fadvertise-us\u002F",{"title":17,"slug":18,"path":19},"Privacy Policy","privacy-policy","\u002Fprivacy-policy\u002F",{"title":21,"slug":22,"path":23},"Abbreviations","abbreviations","\u002Fabbreviations\u002F",{"title":25,"slug":26,"path":27},"Microbes","microbes","\u002Fmicrobes\u002F",{"title":29,"slug":30,"path":31},"Books","recommended-books","\u002Frecommended-books\u002F",{"type":33,"data":34},"blog",{"slug":35,"title":36,"description":37,"seoTitle":38,"seoDescription":38,"author":39,"createdDate":40,"lastUpdatedDate":41,"draft":42,"category":43,"image":38,"body":44,"faq":45,"tags":76,"related":78},"agarose-gel-electrophoresis","Agarose Gel Electrophoresis: Principle, Procedure, Results","Agarose gel electrophoresis separates DNA fragments from 100 bp to 25 kb by size. Learn the principle, how to cast and run a gel, why supercoiled plasmid runs faster than linear DNA, and how to read a PCR gel against a DNA ladder.",null,"Acharya Tankeshwar","2019-09-13","2026-07-17",false,"lab-equipment","You have run a PCR. The tube looks exactly as it did before: clear and colorless. PCR gives you no result you can see. To find out whether the reaction amplified anything, and whether it amplified a product of the right size, you run it on an agarose gel.\n\nLoad four lanes: a DNA ladder, your sample, a positive control, and a negative control. An hour later, under the transilluminator, the gel answers three separate questions at once.\n\n- The **negative control** must be empty. A band here means contamination, and nothing else on the gel can be trusted.\n- The **positive control** must show a band at the expected size. If it does not, the reaction failed, and a blank sample lane means nothing.\n- Only then do you read the **sample lane**, against the **ladder**, which converts the band's height into a number of base pairs.\n\nThat is what an agarose gel does. It turns an invisible mixture of DNA fragments into a pattern you can photograph and measure. Everything below, the agarose percentage, the buffer, the loading dye, the ladder, exists so that the height of a band can be trusted to mean a size.\n\n> **What is agarose gel electrophoresis?**\n>\n> Agarose gel electrophoresis separates DNA fragments by size. DNA carries a negatively charged phosphate backbone, so in an electric field every fragment moves toward the positive electrode (the anode). Because charge and mass increase together along a DNA molecule, all fragments feel the same pull per unit mass, and the only thing separating them is how easily they thread through the pores of the agarose mesh. **Smaller fragments travel further. Larger fragments lag behind.** Migration distance is inversely proportional to the logarithm of fragment size, so a fragment of unknown length can be sized by comparing its position against a DNA ladder run alongside it.\n\nAgarose gel electrophoresis is one of the most common electrophoresis techniques. It is simple to perform and yet has great resolving power. The agarose gel consists of microscopic pores that act as a molecular sieve, separating molecules according to their size and shape as they are drawn through the mesh by an electric field.\n\nIt is used routinely to analyze DNA fragments generated by restriction enzymes or by PCR, and it separates fragments across a range of roughly 100 bp to 25 kb. DNA fragments smaller than 100 bp are better resolved by polyacrylamide gel electrophoresis (PAGE), while fragments larger than 25 kb require pulsed-field gel electrophoresis (PFGE). Agarose gel electrophoresis can also separate other charged biomolecules such as RNA and proteins.\n\n## Principle\n\nThe separation medium is a gel made from agarose. Agarose is isolated from the red seaweed genera Gelidium and Gracilaria. It is a linear polysaccharide built from repeating agarobiose units, each a disaccharide of D-galactose and 3,6-anhydro-L-galactose. During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel’s molecular sieving properties. In general, the higher the concentration of agarose, the smaller the pore size.\n\n![ - Agarose gel electrophoresis experiment overview (Image Source: Ref-2)](https:\u002F\u002Fassets.microbeonline.com\u002Fblogs\u002FAgarose-gel-electrophoresis-experiment-overview.jpg)Figure: Agarose gel electrophoresis experiment overview (Image Source: Ref-2)\n\nTo separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current is applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, **DNA fragments will migrate to the positively charged anode.** Because DNA has a uniform mass\u002Fcharge ratio, DNA molecules are separated by size.\n\n### Factors affecting the migration of DNA\n\n1. **Agarose concentration**\n\nThe mobility of DNA decreases as the agarose concentration rises, because a higher percentage produces a tighter mesh. Higher percentage gels are sturdier and easier to handle, but molecules migrate more slowly through them and staining takes longer to penetrate. The concentration should be matched to the fragment size of interest: a low percentage gel spreads out large fragments, and a high percentage gel resolves small ones. A concentration of 0.8% is a common default for routine DNA separations, while 1% to 2% is used for smaller fragments.\n\n2. **Size of DNA molecule**\n\nThe sieving properties of the agarose gel influence the rate at which a molecule migrates. The separation occurs because smaller molecules pass through the pores of the gel more easily than larger ones. If the size of the two fragments is similar or identical, they will migrate together in the gel.\n\n3. **DNA conformation**\n\nTwo DNA molecules with the same number of base pairs can migrate at different rates if their shapes differ, because the gel sieves by effective size, not by base-pair count.\n\nA single circular plasmid illustrates this well. Extracted plasmid DNA usually exists in three conformations, and on a gel they resolve into three bands even though every molecule contains the same number of base pairs:\n\nSupercoiled plasmid is tightly wound and compact. It has the smallest effective radius and migrates fastest.\n\nLinear DNA, produced when the circle is cut once, migrates at an intermediate rate.\n\nOpen circular (nicked) plasmid is a relaxed floppy loop with the largest effective radius. It snags in the mesh and migrates slowest.\n\nSeeing three bands from one plasmid preparation is therefore normal, and not evidence of contamination.\n\nFor linear fragments, which is what a PCR product or a restriction digest gives you, migration is straightforward: the migration distance is inversely proportional to the log10 of fragment size in base pairs. The smaller the linear fragment, the further it travels.\n\n4. **Applied voltage**\n\nMobility of DNA molecules is also affected by the applied voltage. Within a range, the higher the applied voltage, the faster the sample migration.\n\n## Procedure of Agarose Gel Electrophoresis\n\n### Preparation of Agarose gel matrix\n\nThe centerpiece of agarose gel electrophoresis is the horizontal gel electrophoresis apparatus. The gel is made by dissolving agarose powder in a boiling buffer solution.\n\nThe concentration of agarose in a gel depends on the sizes of the DNA fragments to be separated, with most gels ranging between 0.5%-2%. The solution is then cooled to approximately **55°C** and poured into a casting tray which serves as a mold. A well-former template (often called a comb) is placed across the end of the casting tray to form wells when the gel solution solidifies.\n\n![ - A solidified agarose gel after removal of the comb  (Image Source: Ref-1)](https:\u002F\u002Fassets.microbeonline.com\u002Fblogs\u002FA-solidified-agarose-gel-after-removal-of-the-com.jpg)Figure: A solidified agarose gel after removal of the comb (Image Source: Ref-1)\n\nAfter the gel solidifies, it is submerged in a buffer-filled electrophoresis chamber which contains a positive electrode (anode) at one end and a negative electrode (cathode) at the other. The buffer should cover the gel surface by roughly 2 to 5 mm. Too little buffer allows the gel to dry and overheat. Too much buffer allows current to shunt through the buffer layer above the gel rather than through the gel itself, which generates heat and blurs the bands.\n\nThe two common running buffers are TAE (1X: 40 mM Tris-acetate, 1 mM EDTA) and TBE (0.5X: 45 mM Tris-borate, 1 mM EDTA). They are not interchangeable. TAE has a lower buffering capacity and will exhaust during a long run, but it resolves large fragments well and leaves the DNA clean enough for downstream enzymatic work. TBE has a much higher buffering capacity and gives sharper resolution of small fragments, but borate inhibits many enzymes and carries over into DNA recovered from the gel. As a rule of thumb: use TAE if you intend to cut the band out and use the DNA, and TBE if you only need to look at it.\n\n### Sample preparation and loading\n\nSamples are prepared for electrophoresis by mixing them with loading dyes. Gel loading dye is typically supplied at 6X concentration (0.25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol). Loading dyes used in gel electrophoresis serve three major purposes:\n\n1. add density to the sample, so that it sinks to the bottom of the well rather than drifting out into the running buffer.\n2. provide color and simplify the loading process.\n3. the dyes move at standard rates through the gel, allowing for the estimation of the distance that DNA fragments have migrated.\n\n![ - Loading the DNA sample into a well in the gel  (Image Source: Ref-1)](https:\u002F\u002Fassets.microbeonline.com\u002Fblogs\u002FLoading-the-DNA-sample-into-a-well-in-the-gel.jpg)Figure: Loading the DNA sample into a well in the gel (Image Source: Ref-1)\n\nThese samples are delivered to the sample wells with a clean, adjustable-volume [micropipette](https:\u002F\u002Fmicrobeonline.com\u002Fmicropipette-parts-types-and-uses\u002F).\n\n> Ethidium bromide can be added to the gel during this step or alternatively, the gel may also be stained after electrophoresis in running buffer containing 0.5 μg\u002Fml EtBr for 15-30 min, followed by destaining in running buffer for an equal length of time.\n\n### Applying electric current and separating biomolecules\n\nA direct current (D.C.) power source is connected to the electrophoresis apparatus and an electrical current is applied.  Charged molecules in the sample enter the gel through the walls of the wells. Molecules having **a net negative charge** migrate towards the **positive electrode (anode)** while net **positively charged molecules** migrate towards the **negative electrode (cathode).** The buffer serves as a conductor of electricity and controls the pH, which is important to the charge and stability of biological molecules. Since **DNA has a strong negative** charge at neutral pH, it migrates through the gel towards the **positive electrode** during electrophoresis.\n\nThe bluish-purple dye allows for visual tracking of sample migration during electrophoresis. The gel is run until the dye has migrated to an appropriate distance.\n\n## Results: How to Read an Agarose Gel\n\n### Visualization\n\nThe gel may be stained in either of two ways. Ethidium bromide can be added directly to the molten agarose before casting, so that DNA becomes visible while the run is still in progress. Alternatively, the gel is post-stained after electrophoresis, which gives a cleaner background because unbound dye has not been driven through the gel. The most commonly used stain for visualizing DNA is ethidium bromide (EtBr)\n\nAlternative stains for DNA in agarose gels include SYBR Gold, SYBR Green, crystal violet, and methylene blue. Methylene blue and crystal violet are considerably less sensitive than ethidium bromide, but they are safer to handle and require no ultraviolet light. SYBR Gold and SYBR Green are highly sensitive and much safer than ethidium bromide, but they are more expensive.\n\nEtBr works by intercalating itself in the DNA molecule in a concentration-dependent manner. When exposed to an ultraviolet light source (transilluminator), electrons in the aromatic ring of the ethidium molecule are activated, which leads to the release of energy (light) as the electrons return to the ground state. This allows for an estimation of the amount of DNA in any particular DNA band based on its intensity.\n\n> Ethidium bromide is a suspect mutagen and carcinogen, so it must be handled cautiously. It is hazardous waste and must be disposed of according to local regulations. Stains containing methylene blue are considered safer, but should still be handled and disposed of with care.\n>\n> A second hazard is often overlooked, and it threatens the DNA rather than the operator. Short-wave ultraviolet light (254 nm) nicks DNA and induces thymine dimers. If a band is to be excised from the gel for cloning or sequencing, exposure must be kept to a minimum. Use a long-wave ultraviolet source or a blue-light transilluminator, and cut the band quickly rather than leaving the gel under the lamp.\n\nThe exact **sizes** of separated DNA fragments can be determined by plotting the log of the molecular weight for the different bands of a DNA standard (DNA ladder) against the distance traveled by each band. The DNA standard contains a mixture of DNA fragments of pre-determined sizes that can be compared against the unknown DNA samples.\n\n![ - An image of a gel post electrophoresis  (Image Source: Ref-1)](https:\u002F\u002Fassets.microbeonline.com\u002Fblogs\u002FAgarose-gel-electrophoresis-reading.png)Figure: An image of a gel post electrophoresis (Image Source: Ref-1)\n\nDNA concentrations can be estimated by\n\nA. Taking absorbance at 260 nm. At 260 nm, an absorbance (A) of 1 unit corresponds to a concentration of:\n\n- 50 μg\u002Fml for dsDNA\n- 40 μg\u002Fml for RNA\n- 33 μg\u002Fml for ssDNA\n- 20-30 µg\u002Fml for oligonucleotides\n\nAlthough this method is quick and nondestructive and gives information about the purity of the sample (e.g., presence of protein or organic contaminants), reliable estimates are obtained only with concentrations of at least 1 μg\u002Fml. Additionally, this method cannot distinguish between DNA and RNA.\n\n**B. Intensity of Ethidium Bromide Fluorescence:**\n\nThe amount of DNA in a sample can **be estimated from the intensity of ethidium bromide fluorescence** (fluorescence emitted by ethidium bromide is proportional to the amount of DNA). The DNA quantity in an “unknown” solution can be estimated by comparing its level of fluorescence with the intensity of known amounts of DNA of similar size. This method is useful if a DNA sample is contaminated with other compounds that absorb in the UV range or is too dilute to measure at 260 nm.\n\n## Reading a diagnostic PCR gel\n\nA typical diagnostic gel carries four lanes, and each answers a different question.\n\n**The ladder** is a mixture of fragments of known size, run alongside the samples. It converts height into base pairs. Without it, a band is just a band.\n\n**The negative control** contains everything except template DNA. It must be **empty**. A band here means the reagents, the pipettes, or the air of the room carried contaminating DNA, and every other lane on the gel is now uninterpretable. This lane is checked first, before anyone looks at the patient.\n\n**The positive control** contains known target DNA. It must show a band at the expected size. If it does not, the reaction failed, and a blank patient lane means nothing at all. An absent band is only a negative result when the positive control worked.\n\n**The patient lane** is read last, and only against the other three.\n\n**What the common patterns mean:**\n\n| What you see | What it usually means |\n| --- | --- |\n| Sharp band at the expected size | Target amplified. The result you wanted |\n| No band anywhere, including the positive control | Reaction failed. Check reagents, cycling, or the DNA template |\n| No band in the patient lane, positive control good | A true negative for that target |\n| Band in the negative control | Contamination. Discard the run, decontaminate, repeat |\n| Band at the wrong size | Non-specific amplification. Primers annealed somewhere unintended |\n| Multiple faint bands or a ladder-like smear of products | Non-specific priming. Raise the annealing temperature |\n| A continuous smear in the sample lane | Degraded DNA, or gross overloading of the well |\n| Bands curving or \"smiling\" across the gel | The gel overheated. Lower the voltage |\n\n**A note on the dyes.** Loading dye is not just for colour. In a 1% agarose gel, bromophenol blue migrates at roughly the position of a 300 to 500 bp fragment, and xylene cyanol at roughly 4 kb. If your amplicon is 250 bp and the bromophenol blue front has reached the end of the gel, your product has already run off. Watch the dye, not the clock.\n\n## Key exam facts in one table\n\n| Concept | Fact to retain |\n| --- | --- |\n| What agarose is | A linear polysaccharide from red seaweed (*Gelidium*, *Gracilaria*), built from repeating **agarobiose** units of D-galactose and 3,6-anhydro-L-galactose |\n| Gel formation | Agarose is dissolved in boiling buffer, cooled to \\~55°C, and poured. Polymers associate non-covalently into a bundled network whose pores act as a molecular sieve |\n| Orientation | **Horizontal** (\"submarine\") gel, submerged in buffer. Contrast: PAGE is vertical |\n| Direction of DNA migration | Toward the **anode** (positive electrode). The phosphate backbone is negatively charged at all working pH |\n| Why size, not charge | DNA has a **uniform charge-to-mass ratio**. Charge sets direction; the sieving matrix sets separation |\n| Migration and size | Migration distance is inversely proportional to **log₁₀** of fragment size in base pairs |\n| Agarose concentration | Higher % = smaller pores = better resolution of **small** fragments. Typical range 0.5% to 2%; 0.8% is a common default |\n| Resolving range | Roughly **100 bp to 25 kb**. Below this, use **PAGE**. Above this, use **PFGE** |\n| Conformation order | **Supercoiled (fastest) > linear > open circular\u002Fnicked (slowest)** for the same plasmid |\n| Running buffers | **TAE** (Tris-acetate-EDTA) and **TBE** (Tris-borate-EDTA) |\n| TAE vs TBE | TAE: lower buffering capacity, better for large fragments, DNA recoverable for enzymatic work. TBE: higher buffering capacity, sharper small fragments, but borate inhibits enzymes |\n| Buffer depth | Should cover the gel by about 2 to 5 mm. Excess buffer shunts current above the gel, causing heating and band blurring |\n| Loading dye | Adds glycerol (density, so the sample sinks into the **well**), colour, and tracking dyes |\n| Tracking dyes | **Bromophenol blue** ≈ 300 to 500 bp; **xylene cyanol** ≈ 4 kb, in a 1% gel |\n| Standard stain | **Ethidium bromide** (0.5 µg\u002Fml), an intercalating dye. Fluoresces under UV. A suspect mutagen and carcinogen |\n| Safer stains | SYBR Gold, SYBR Green (sensitive, expensive); methylene blue, crystal violet (safer, less sensitive) |\n| UV hazard to DNA | Short-wave UV nicks and dimerizes DNA. Use long-wave UV or a blue-light transilluminator if the band will be excised for cloning |\n| A₂₆₀ = 1.0 corresponds to | 50 µg\u002Fml dsDNA; 40 µg\u002Fml RNA; 33 µg\u002Fml ssDNA; 20 to 30 µg\u002Fml oligonucleotides |\n| Reading a gel | Negative control must be **empty**. Positive control must show a band. Only then is the patient lane interpretable |\n| Key clinical use | Sizing a PCR amplicon against a ladder to confirm a pathogen when culture is sterile, for example after prior antibiotic therapy |\n\n## Where Students Get Confused\n\n**\"Does agarose gel electrophoresis separate DNA by charge or by size?\"** By size. This trips people up because the whole thing runs on charge. Charge is what *moves* the DNA, and it moves every fragment toward the anode with the same force per unit mass, because adding a base pair adds charge and mass in fixed proportion. Charge therefore separates nothing. The gel mesh separates. If a question asks what agarose gel electrophoresis separates DNA by, the answer is size (and conformation), not charge.\n\n**\"Why does my plasmid prep show three bands? Is it contaminated?\"** Almost certainly not. One plasmid can exist in three conformations, and they run at three different heights despite having identical base-pair counts. **Supercoiled** is tightly wound and compact, so it slips through the mesh fastest. **Open circular (nicked)** is a relaxed floppy loop with a large effective radius, and it snags, so it runs slowest. **Linear** sits in between. Three bands, one plasmid.\n\n**\"So a higher percentage gel is a better gel?\"** Only for smaller fragments. Percentage sets pore size, and pore size should match your target. A 2% gel has a tight mesh that resolves a 200 bp fragment beautifully and leaves a 10 kb fragment stuck near the well. A 0.5% gel does the opposite. Choose the percentage from the fragment size, never the other way round.\n\n**\"My PCR gel is blank. Is the patient negative?\"** You do not know yet. A blank patient lane is only a negative result if the **positive control** produced a band. If the positive control is also blank, the reaction failed, and the patient's lane carries no information. Equally, if the **negative control** shows a band, the run is contaminated and even a beautiful patient band cannot be trusted. Read the controls first, always.\n\n**\"TAE or TBE, does it matter?\"** Yes, and the deciding question is what happens to the DNA next. If you are going to cut the band out of the gel and ligate, sequence, or digest it, use **TAE**, because borate from TBE carries over and inhibits enzymes. If you only need to look at the gel, especially at small fragments over a long run, use **TBE**, whose higher buffering capacity resists exhaustion and gives sharper small bands.\n\n**\"The dye sinks into the gel, right?\"** It sinks into the **well**. Glycerol in the loading dye makes the sample denser than the running buffer above it, so the sample settles to the bottom of the well instead of drifting away into the tank. The gel is a solid. Nothing sinks into it.\n\n**\"I cut my band out under UV and the cloning never works.\"** Short-wave ultraviolet light nicks DNA and creates thymine dimers. The longer the gel sits on a 254 nm transilluminator, the more damaged the fragment you are about to clone. Use a long-wave UV source or a blue-light transilluminator, cut fast, and do not leave the gel sitting under the lamp while you find the scalpel.\n\n**\"Why is my gel smiling?\"** Bands that curve upward at the edges mean the gel overheated during the run, usually because the voltage was too high or the buffer was exhausted or too deep. Heat is generated in proportion to current. Lower the voltage and accept a longer run.\n\n**References and further reading**\n\n1. Lee PY, Costumbrado J, Hsu CY, Kim YH. Agarose gel electrophoresis for the separation of DNA fragments. Journal of Visualized Experiments. 2012;(62):3923. doi:10.3791\u002F3923\n2. Green MR, Sambrook J. Molecular Cloning: A Laboratory Manual. 4th ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 2012.\n3. Wilson K, Walker J. Principles and Techniques of Biochemistry and Molecular Biology. 8th ed. Cambridge: Cambridge University Press; 2018. Chapter: Electrophoretic Techniques.\n4. Westermeier R. Electrophoresis in Practice: A Guide to Methods and Applications of DNA and Protein Separations. 5th ed. Weinheim: Wiley-VCH; 2016.",[46,49,52,55,58,61,64,67,70,73],{"question":47,"answer":48},"Does agarose gel electrophoresis separate DNA by size or by charge?","By size. Charge determines the direction of travel, because DNA's phosphate backbone is negatively charged and every fragment therefore migrates toward the anode. But DNA has a uniform charge-to-mass ratio, so every fragment experiences the same pull per unit mass, and charge separates nothing. The sieving action of the agarose mesh does all the separating, which is why migration distance reports fragment size.",{"question":50,"answer":51},"Why does DNA move toward the anode?","The phosphate backbone of DNA carries a negative charge at every pH used in the laboratory, making DNA an anion. Anions are attracted to the positive electrode, which is the anode. Unlike proteins, DNA has no isoelectric point to consider, so it always migrates in the same direction.",{"question":53,"answer":54},"What size range can agarose gel electrophoresis resolve?","Roughly 100 base pairs to 25 kilobases, depending on the agarose concentration. Fragments smaller than about 100 bp are better resolved by polyacrylamide gel electrophoresis, and fragments larger than about 25 kb require pulsed-field gel electrophoresis.",{"question":56,"answer":57},"Why does my plasmid preparation show three bands on the gel?","Because a circular plasmid exists in three conformations, and the gel separates by effective size rather than base-pair count. Supercoiled plasmid is tightly wound and compact, so it migrates fastest. Open circular (nicked) plasmid is a relaxed floppy loop with a large effective radius, so it snags in the mesh and migrates slowest. Linear plasmid runs in between. All three contain the same number of base pairs. Three bands from one preparation is normal, not a sign of contamination.",{"question":59,"answer":60},"How do I choose the agarose concentration?","Match the pore size to the fragment size. A low-percentage gel (around 0.5 to 0.8%) has large pores and resolves large fragments, while small fragments run through almost unimpeded. A high-percentage gel (1.5 to 2%) has small pores that resolve small fragments sharply while holding large fragments near the well. Higher percentage does not mean better resolution in general, only better resolution of smaller fragments.",{"question":62,"answer":63},"What is the difference between TAE and TBE buffer?","TAE (Tris-acetate-EDTA) has a lower buffering capacity and will exhaust during long runs, but it resolves large fragments well and the DNA recovered from a TAE gel is clean enough for downstream enzymatic work. TBE (Tris-borate-EDTA) has a much higher buffering capacity and gives sharper resolution of small fragments, but borate inhibits many enzymes and carries over into extracted DNA. Use TAE if you plan to cut the band out and use the DNA, and TBE if you only need to visualize it.",{"question":65,"answer":66},"What is a DNA ladder and why is it needed?","A DNA ladder is a mixture of DNA fragments of known sizes, run in a lane alongside the samples. It converts the height of a band into a number of base pairs. Without a ladder, the position of a band carries no information, because migration distance depends on the gel percentage, the voltage, and the run time.",{"question":68,"answer":69},"My PCR gel shows no band in the patient lane. Is that a negative result?","Only if the positive control produced a band. If the positive control is also blank, the reaction itself failed and the patient's lane carries no information. Likewise, if the negative control shows a band, the run is contaminated and no lane on that gel can be trusted. The controls are always read before the patient's sample.",{"question":71,"answer":72},"What do the dyes in the loading buffer do?","Loading dye serves three purposes. Glycerol makes the sample dense enough to sink to the bottom of the well instead of drifting into the buffer. The colour makes loading easier to see. And the tracking dyes migrate at predictable rates, marking how far the run has progressed. In a 1% agarose gel, bromophenol blue migrates at approximately the position of a 300 to 500 bp fragment and xylene cyanol at approximately 4 kb.",{"question":74,"answer":75},"Is ethidium bromide dangerous, and what can be used instead?","Ethidium bromide intercalates into DNA and is a suspect mutagen and carcinogen, so it requires gloves and regulated disposal. Safer alternatives include SYBR Gold and SYBR Green, which are highly sensitive but expensive, and methylene blue or crystal violet, which are much safer but considerably less sensitive. A separate hazard is the short-wave ultraviolet light used to visualize ethidium bromide, which nicks DNA. If the band is to be excised for cloning, use a long-wave ultraviolet or blue-light transilluminator.",[77],"electrophoresis",[79,118,156,163,199,206],{"slug":80,"title":81,"description":82,"seoTitle":38,"seoDescription":38,"author":83,"createdDate":84,"lastUpdatedDate":85,"draft":42,"category":43,"image":38,"faq":86,"tags":117},"polyacrylamide-gel-electrophoresis-page","Polyacrylamide Gel Electrophoresis (PAGE): Principle and Procedure","Polyacrylamide gel electrophoresis (PAGE) separates proteins by size. Learn why SDS is added, why the gel has a stacking and a resolving layer, how to choose the acrylamide percentage, and how SDS-PAGE underpins the Western blot.","Samikshya Acharya","2023-03-19","2026-07-11",[87,90,93,96,99,102,105,108,111,114],{"question":88,"answer":89},"Why is SDS added in SDS-PAGE?","SDS is an anionic detergent that unfolds the protein and binds along the polypeptide chain at a roughly constant ratio of about one SDS molecule per two amino acid residues. This gives every protein a negative charge proportional to its length, so that charge per unit mass becomes the same for all proteins. With the charge variable removed, migration depends on size alone, and the distance a band travels can be read directly as a molecular weight.",{"question":91,"answer":92},"Why does an SDS-PAGE gel have two layers?","The upper stacking gel (pH 6.8, large pores) does no separating. Its job is to compress proteins scattered throughout the depth of the loading well into a single thin disc, using a discontinuous buffer system in which slow-moving glycine trails and fast-moving chloride leads, sandwiching the proteins between them. When the disc reaches the lower resolving gel (pH 8.8, small pores), glycine ionizes and overtakes, the sandwich collapses, and all proteins begin separating from the same starting line. Without a stacking gel, every band would be a smear.",{"question":94,"answer":95},"What is the difference between native PAGE and SDS-PAGE?","SDS-PAGE denatures the protein with SDS and a reducing agent, so separation is by size alone and the protein is no longer functional. Native PAGE uses neither, so the protein retains its fold, subunits, and intrinsic charge, and separation depends on charge, size, and shape together. Use SDS-PAGE to measure the size of a polypeptide chain, and native PAGE to study a protein that must remain active or intact.",{"question":97,"answer":98},"How do I choose the acrylamide percentage?","Match the pore size to the size of your target. A low-percentage gel (4 to 8%) has large pores and resolves large proteins, while small proteins run straight through. A high-percentage gel (12 to 20%) has small pores that resolve small proteins sharply while large proteins barely enter the gel. Higher percentage does not mean better resolution in general; it means better resolution of smaller molecules.",{"question":100,"answer":101},"Which direction do proteins move in SDS-PAGE, and why?","Toward the anode, the positive electrode. SDS coats every protein with a strong negative charge, so all proteins become anions and are attracted to the positive electrode. This is why SDS-PAGE gels are run vertically with the anode at the bottom.",{"question":103,"answer":104},"What is the role of β-mercaptoethanol, and how is it different from SDS?","They denature different things. SDS unfolds the polypeptide chain and coats it with charge, but it cannot break covalent disulfide bonds. β-mercaptoethanol is a reducing agent that cleaves those bonds, separating proteins into their individual polypeptide chains. Immunoglobulin G, for example, runs as a single band of about 150 kDa without a reducing agent, and splits into heavy chains of about 50 kDa and light chains of about 25 kDa when β-mercaptoethanol is added.",{"question":106,"answer":107},"What do APS and TEMED do?","Ammonium persulfate (APS) is the free-radical initiator that starts acrylamide polymerization, and TEMED is the catalyst that accelerates radical formation from APS. Both are added immediately before the gel is poured, because polymerization begins as soon as they are mixed in. Oxygen inhibits polymerization, which is why water-saturated isobutanol is layered over the resolving gel to exclude air.",{"question":109,"answer":110},"Is polyacrylamide gel toxic?","Unpolymerized acrylamide monomer is a potent neurotoxin and a probable human carcinogen, and it is absorbed through the skin, so the powder and the unset gel solution must be handled with gloves. Once polymerized, the gel itself is far less hazardous, but it may contain traces of residual monomer, so gloves are worn when handling gels as well.",{"question":112,"answer":113},"What is the tracking dye in SDS-PAGE?","Bromophenol blue, not bromothymol blue. It is a small, fast-migrating dye that runs ahead of nearly all proteins, marking the dye front. It does not stain the proteins. When the dye front approaches the bottom of the gel, the run is stopped so that the smallest proteins do not run off the end.",{"question":115,"answer":116},"How is SDS-PAGE related to the Western blot?","SDS-PAGE is the first step of a Western blot. Proteins are separated by molecular weight on the gel, transferred to a membrane, and then probed with antibodies. Because separation is by size alone, the position of a band on the membrane identifies the protein. This is why HIV proteins carry names such as p24, gp41, and gp120: the numbers are the molecular weights in kilodaltons at which those proteins resolve.",[77],{"slug":119,"title":120,"description":121,"seoTitle":38,"seoDescription":38,"author":122,"createdDate":123,"lastUpdatedDate":85,"draft":42,"category":43,"image":38,"faq":124,"tags":155},"cellulose-acetate-electrophoresis","Cellulose Acetate Electrophoresis: Principle and Application","Cellulose acetate electrophoresis separates serum proteins and hemoglobin variants by net charge at alkaline pH. Learn the principle, why Hb S co-migrates with Hb D and Hb G, and why a sickle cell result must be confirmed at acid pH.","Ashma Shrestha","2022-12-25",[125,128,131,134,137,140,143,146,149,152],{"question":126,"answer":127},"What is the principle of cellulose acetate electrophoresis?","Proteins are separated on a cellulose acetate membrane soaked in an alkaline buffer, usually pH 8.4 to 8.6. Because the membrane's pores are large compared with a protein, it does almost no sieving, so separation depends on net charge rather than on size. At this alkaline pH the buffer is above the isoelectric point of every serum protein, so all carry a net negative charge and migrate toward the anode. The greater the net negative charge, the further the protein travels.",{"question":129,"answer":130},"Does cellulose acetate electrophoresis separate proteins by their isoelectric point?","No. A protein at its isoelectric point carries no net charge and does not migrate at all. Separation occurs because the buffer pH is held above the isoelectric point of every protein, so all are negatively charged and all move. The isoelectric point determines how negatively charged a protein is at the working pH, and therefore how fast it travels, but the separation does not occur at the isoelectric point. Separating molecules at their isoelectric points is a different technique, called isoelectric focusing.",{"question":132,"answer":133},"Why is cellulose acetate used for hemoglobin rather than a gel?","Hemoglobin variants such as Hb A and Hb S differ by a single amino acid and are essentially identical in size. A sieving matrix such as agarose or polyacrylamide separates by size and therefore cannot distinguish them. The large pores of cellulose acetate mean the membrane contributes almost nothing mechanically, so the separation is driven purely by the charge difference that the amino acid substitution creates.",{"question":135,"answer":136},"In what order do hemoglobins migrate on alkaline cellulose acetate?","Toward the anode, fastest first: Hb A, then Hb F, then Hb S, then Hb C. In Hb S, the negatively charged glutamate at position 6 of the beta chain is replaced by a neutral valine, reducing the net negative charge, so Hb S lags behind Hb A. In Hb C that same glutamate is replaced by a positively charged lysine, so Hb C lags further still.",{"question":138,"answer":139},"Can sickle cell disease be diagnosed from an alkaline cellulose acetate strip alone?","No. At alkaline pH, Hb S co-migrates with Hb D, Hb G, and Hb Lepore, and Hb C co-migrates with Hb E, Hb O-Arab, and Hb A2. A band at the S position identifies a charge, not a hemoglobin. Confirmation requires electrophoresis on citrate agar at acid pH, around 6.0 to 6.2, where Hb S separates from Hb D and Hb G, supported by a sickle solubility test.",{"question":141,"answer":142},"How do you distinguish sickle cell trait from sickle cell disease on the strip?","Look for the Hb A band. Sickle cell trait shows two bands, with Hb A denser than Hb S in roughly a 60 to 40 ratio. Sickle cell disease shows no Hb A band at all, a dense Hb S band, and often a raised Hb F. The absence of Hb A is what makes the diagnosis. A recent blood transfusion can introduce a donor-derived Hb A band and obscure this.",{"question":144,"answer":145},"Why are newborn screening results written as FS or FAS?","Hemoglobin bands in newborn screening are reported in descending order of abundance, and a newborn still produces mostly fetal hemoglobin. FA is a normal newborn, FAS indicates sickle cell trait, and FS indicates sickle cell disease, because no Hb A is present.",{"question":147,"answer":148},"Why do gamma globulins appear behind the point of application?","Because of electroendosmosis. Fixed negative charges on the cellulose acetate attract cations from the buffer, and the resulting bulk flow of buffer moves toward the cathode. This flow opposes the anodal migration of the proteins. Albumin carries enough net charge to overcome it easily, but the gamma globulins, which are the least negatively charged fraction, are dragged back to or slightly past the origin.",{"question":150,"answer":151},"How are the bands visualized on a cellulose acetate strip?","The strip is stained with a protein dye such as Ponceau S, Amido Black, or Coomassie brilliant blue, then destained in dilute acetic acid to remove background. It is then cleared in a methanol or acetic acid mixture, which turns the cellulose acetate optically transparent, allowing the strip to be scanned directly in a densitometer for quantification and stored as a permanent record. Where the analytes are enzymes, a zymogram may be used instead, revealing bands by their enzymatic activity.",{"question":153,"answer":154},"Is cellulose acetate electrophoresis still used?","Yes. In well-resourced laboratories, HPLC and capillary electrophoresis have largely replaced it for hemoglobin variant analysis, because they are automated, quantitative, and resolve variants that co-migrate at alkaline pH. Cellulose acetate remains widely used where those instruments are unavailable, and it retains the advantages of being simple, rapid, and inexpensive.",[77],{"slug":157,"title":158,"description":158,"seoTitle":38,"seoDescription":38,"author":122,"createdDate":159,"lastUpdatedDate":160,"draft":42,"category":43,"image":38,"faq":161,"tags":162},"capillary-electrophoresis","Capillary Electrophoresis: Principle and Application","2022-12-16","2026-07-05",[],[77],{"slug":164,"title":165,"description":166,"seoTitle":38,"seoDescription":38,"author":167,"createdDate":168,"lastUpdatedDate":169,"draft":42,"category":43,"image":38,"faq":170,"tags":198},"electrophoresis-principles-types-and-uses","Electrophoresis: Principles, Types, and Uses","Electrophoresis separates charged molecules such as proteins and DNA by moving them through a gel in an electric field. Learn the principle, the factors that control mobility, the main types, and how serum protein electrophoresis detects multiple myeloma.","Srijana Khanal","2022-07-13","2026-07-10",[171,174,177,180,183,186,189,192,195],{"question":172,"answer":173},"What is the basic principle of electrophoresis?","Charged molecules placed in an electric field migrate toward the electrode of opposite charge. Negatively charged molecules (anions) move toward the positive anode, and positively charged molecules (cations) move toward the negative cathode. Each molecule travels at a speed set by its electrophoretic mobility, which depends on its net charge, its size and shape, and the viscosity and pore size of the medium. Molecules separate only if their mobilities differ.",{"question":175,"answer":176},"Why does DNA always move toward the anode?","DNA carries a phosphate backbone that remains negatively charged at any pH used in the laboratory. Because it is always an anion, it is always attracted to the positive anode. Its charge-to-mass ratio is also nearly constant regardless of fragment length, which is why DNA fragments separate essentially by size alone.",{"question":178,"answer":179},"Which way does a protein move in electrophoresis?","It depends on the buffer pH relative to the protein's isoelectric point (pI). Above its pI the protein is net negative and moves toward the anode. Below its pI it is net positive and moves toward the cathode. At exactly its pI, its net charge is zero and it does not migrate.",{"question":181,"answer":182},"Why is electrophoresis called an incomplete form of electrolysis?","In electrolysis, ions travel all the way to the electrode and undergo discharge there. In electrophoresis the electric field is switched off while the molecules are still in transit, so they never reach the electrode. What matters is not the reaction at the electrode but how far each molecule traveled, because that distance is the separation.",{"question":184,"answer":185},"What is the difference between zone and moving boundary electrophoresis?","In zone electrophoresis the sample is applied as a narrow zone on a supporting medium such as paper, cellulose acetate, or a gel, and components resolve into discrete bands. In moving boundary electrophoresis the separation occurs in free solution with no supporting medium, and the components appear as moving boundaries rather than distinct bands. The classical example of the latter is the Tiselius apparatus.",{"question":187,"answer":188},"What are the main factors affecting electrophoretic mobility?","Inherent factors include the net charge of the molecule, its charge density, its molecular weight, and its size and shape. External factors include the applied voltage, current and power, the pore size and viscosity of the supporting medium, the temperature, and the pH of the buffer, which determines the net charge on ampholytes such as proteins.",{"question":190,"answer":191},"How is electrophoresis used to diagnose multiple myeloma?","Serum protein electrophoresis separates serum proteins into albumin and the alpha, beta, and gamma globulin fractions. Normal gamma globulins are produced by thousands of plasma cell clones with slightly different mobilities, so they form a broad band. In multiple myeloma a single malignant clone produces one identical immunoglobulin, and these identical molecules migrate together to produce a sharp, narrow monoclonal (M) band in the gamma region.",{"question":193,"answer":194},"Does electrophoresis separate molecules by size or by charge?","By both, because mobility depends on the ratio of net charge to size. SDS-PAGE deliberately removes the charge variable by coating every protein with a uniform negative charge proportional to its length, so that separation depends on size alone. Native gels, in contrast, separate molecules on the basis of charge and size together.",{"question":196,"answer":197},"Why is a larger pore size not always better?","Larger pores impede migration less, so molecules travel faster, but small molecules pass through almost unhindered and are therefore poorly resolved. The gel concentration is chosen to match the size range of interest: a low-percentage gel resolves large fragments, and a high-percentage gel resolves small ones.",[77],{"slug":200,"title":201,"description":202,"seoTitle":38,"seoDescription":38,"author":39,"createdDate":203,"lastUpdatedDate":85,"draft":42,"category":43,"image":38,"faq":204,"tags":205},"pulsed-field-gel-electrophoresis-pfge","Pulsed-Field Gel Electrophoresis (PFGE): Steps, Applications","Pulsed-field gel electrophoresis (PFGE) separates DNA fragments up to 10 Mb by switching the electric field between directions, forcing large molecules to reorient. Learn why pulsing works, the plug-based steps, and why PFGE was the gold standard for outbreak fingerprinting.","2019-09-16",[],[77],{"slug":207,"title":208,"description":209,"seoTitle":38,"seoDescription":38,"author":39,"createdDate":210,"lastUpdatedDate":85,"draft":42,"category":43,"image":38,"faq":211,"tags":236},"serologic-methods-counterimmunoelectrophoresis-cie","Counterimmunoelectrophoresis (CIE): Principle, Procedure, Uses","Counterimmunoelectrophoresis (CIE) drives antigen and antibody toward each other in an agarose gel, producing a precipitin line within an hour. Learn the principle, why a neutral antibody migrates at all, the procedure, and why pneumococcal serotypes 7 and 14 give false negatives.","2012-09-29",[212,215,218,221,224,227,230,233],{"question":213,"answer":214},"What is the principle of counterimmunoelectrophoresis?","Antigen and antibody are driven toward each other through an agarose gel in an alkaline buffer, and where they meet in optimal proportions they form a visible precipitin line. They move for different reasons. Bacterial capsular antigens are acidic, so they carry a net negative charge at pH 8.4 and migrate toward the anode. Antibodies carry almost no net charge at that pH, but they are swept toward the cathode by electroendosmosis, the bulk flow of buffer through the negatively charged agarose. The result is that the two travel in opposite directions along the same line and collide between the wells.",{"question":216,"answer":217},"If antibodies are electrically neutral, how do they move in CIE?","They do not move because of the electric field acting on them directly. They move because the liquid inside the gel is moving. Agarose carries fixed negative charges that attract cations from the buffer. When the current is applied, those cations migrate toward the cathode and drag hydrating water with them, so the whole buffer phase flows cathodally. This bulk flow, called electroendosmosis, carries the near-neutral antibody toward the cathode. The strongly negative antigen swims against this flow and still reaches the anode.",{"question":219,"answer":220},"Why is it called counterimmunoelectrophoresis?","Because the antigen and antibody migrate counter to one another, in opposite directions along the same axis, so that they are forced to meet. In ordinary electrophoresis everything in the gel migrates in the same direction. An older name for the technique, immunoelectroosmophoresis, describes the mechanism more literally, since electroosmosis is what moves the antibody.",{"question":222,"answer":223},"Which well should the antigen go into?","The cathodal well. The antigen migrates toward the anode, so it must start on the cathodal side to have somewhere to travel. The antibody is carried toward the cathode, so it must start in the anodal well. Each reactant begins at the electrode it is moving away from. If the wells are loaded the other way round, the two reactants migrate apart and no precipitin line can form regardless of how much antigen is present.",{"question":225,"answer":226},"How is CIE different from the Ouchterlony method?","The chemistry is identical. Both rely on antigen and antibody meeting in optimal proportions to form a precipitin line in agar. The difference is that Ouchterlony relies on passive diffusion, which sends the reactants outward in all directions and takes twenty-four to forty-eight hours. CIE applies an electric current that drives them straight toward each other along one line, giving a result in thirty to sixty minutes and detecting roughly ten times less antigen.",{"question":228,"answer":229},"How is counterimmunoelectrophoresis different from immunoelectrophoresis?","They are separate techniques with confusingly similar names. In classical immunoelectrophoresis, described by Grabar and Williams, a protein mixture is first separated by electrophoresis, and only afterwards is antiserum allowed to diffuse passively from a trough, producing precipitin arcs. Electrophoresis and immunodiffusion happen one after the other. In counterimmunoelectrophoresis they happen simultaneously, and the electric field brings the reactants together rather than separating them.",{"question":231,"answer":232},"Why detect capsular antigen rather than culture the organism?","Because capsular polysaccharide persists in cerebrospinal fluid long after antibiotics have killed the bacterium that shed it. A child given a dose of antibiotic before the lumbar puncture may have a blank Gram stain and a sterile culture while the CSF still contains abundant antigen. Antigen detection was developed for exactly this situation, and it delivers an answer in an hour rather than two days.",{"question":234,"answer":235},"Is counterimmunoelectrophoresis still used today?","Rarely in routine diagnostics. Latex particle agglutination is faster, simpler, and more sensitive, and needs no apparatus. For Cryptococcus neoformans, the cryptococcal antigen lateral flow assay is now the recommended method. For bacterial meningitis, multiplex PCR detects the organism's nucleic acid rather than its shed capsule, with far greater sensitivity. CIE survives in teaching, in some reference and veterinary laboratories, and where reagents are inexpensive and the apparatus is already available.",[77,237],"immunoassays",[239,245,251,256,259,263,268,273,276,280],{"slug":240,"name":39,"description":241,"image":242,"body":243,"postCount":244},"acharya-tankeshwar","Editor-in-chief","https:\u002F\u002Fassets.microbeonline.com\u002Fauthors\u002Ftankeshwar-acharya-author-microbeonline.jpg","***Tankeshwar Acharya, MSc (Medical Microbiology)***\n\n*Tankeshwar Acharya is an Assistant Professor in the Department of Microbiology at Patan Academy of Health Sciences (PAHS), Nepal, where he has been teaching and practicing clinical microbiology for over 14 years. He is the founder of Microbe Online, one of the leading free microbiology education resources on the web, covering bacteriology, mycology, parasitology, immunology, and clinical laboratory diagnostics written from direct experience in both the classroom and the diagnostic laboratory.*",433,{"slug":246,"name":122,"description":247,"image":248,"body":249,"postCount":250},"ashma-shrestha","SEO Copywriter and Science Communicator\nKathmandu, Nepal","https:\u002F\u002Fassets.microbeonline.com\u002Fauthors\u002Fashma-shrestha.png","Ashma Shrestha holds a Master of Science in Medical Microbiology from the Institute of Science and Technology (IOST), Tribhuvan University, Nepal, where she developed a strong foundation in virology, molecular biology, and diagnostic microbiology.\n\nShe now works as an SEO Copywriter at Resolution Digital, where she combines her scientific training with research-driven content strategy. She is certified in Google Analytics and Google Business Profile (GBP), and brings a data-informed approach to science communication writing content that is not only accurate but structured to reach and serve the students who need it most.\n\nAt microbeonline, Ashma contributes articles primarily in virology and molecular biology, areas she finds most compelling for their mechanistic depth and their growing clinical relevance. Her writing reflects the same standard the site is built on: factual rigor, clear explanation of the *why* behind microbiology concepts, and content that helps students move from memorization to genuine understanding.\n\nShe is passionate about making complex microbiological concepts accessible without sacrificing accuracy; a skill that sits at the intersection of her scientific training and her professional work in content and SEO.",81,{"slug":252,"name":253,"description":254,"image":38,"body":38,"postCount":255},"sushmita-baniya","Sushmita Baniya","Author \u002F Contributor",32,{"slug":257,"name":83,"description":254,"image":38,"body":38,"postCount":258},"samikshya-acharya",20,{"slug":260,"name":261,"description":254,"image":38,"body":38,"postCount":262},"alisha-tripathi","Alisha Tripathi",6,{"slug":264,"name":265,"description":266,"image":38,"body":38,"postCount":267},"aastha-shrestha","Aastha Shrestha"," Author \u002F Contributor",10,{"slug":269,"name":270,"description":271,"image":38,"body":38,"postCount":272},"guest-author","Guest Author","Guest Author \u002F Contributor",2,{"slug":274,"name":167,"description":254,"image":38,"body":38,"postCount":275},"srijana-khanal",18,{"slug":277,"name":278,"description":271,"image":38,"body":38,"postCount":279},"dr-poonam-acharya","Dr. Poonam Acharya",1,{"slug":281,"name":282,"description":254,"image":38,"body":283,"postCount":284},"nisha-rijal","Nisha Rijal","**Nisha Rijal** is a microbiologist and quality assurance specialist. She served for nearly 12 years as a microbiologist at the National Public Health Laboratory (NPHL), Nepal's national reference laboratory, and continues to work as a consultant microbiologist in international public health organization. ",51]