We are having trouble with agarose gel electrophoresis. It used to work a couple of months ago but now the ladder always look smeared. We switched the components (1x TBE, 100bp ladder, different type of agarose and voltage) and equipment. We also tried 1.2%, 1%, 1.5% and 2% agarose.
I would appreciate it if someone can help us.
Are you sure that your TBE is at the right dilution? And that you're using TBE to make the gel and also as the buffer for running? That sort of wavy line looks like what you might get if you'd made the gel with water instead of buffer, or run it with water instead of buffer. You could also try using TAE to make/run the gel as a sanity check.
Try going even higher with the agarose concentration, as it looks like you have decent separation of the small bands and poor separation of the large bands. Also try a fresh batch of agarose.
I would suggest trying 2-4% in 0.5% steps
If you are running 100bp or smaller fragments, you should in the range of 4% agarose.
Here is a nice example of agarose ranges and minimum difference of PCR products that can be resolved. https://www.qiagen.com/mx/resources/faq?id=1b23a0b3-32b4-4c1b-bd84-49b4b6a58311&lang=en
8.5: Lab Procedures- PCR and Gel Electrophoresis
Polymerase chain reaction (PCR) is molecular technique used to amplify specific regions of DNA for applications such as sequencing and genetic analysis. Typically, there is a limited amount of DNA in the sample to study and amplification is required. PCR is carried out in a test tube with the DNA template, primers specific for the region that is desired, DNA polymerase, and reagents that stabilize the reaction. Once the reaction is put together, it will go into a thermocycler (PCR machine) that will create the conditions for DNA replication to occur. Each round of PCR requires three steps, denaturation, annealing, and elongation, each of which doubles the amount of DNA template present in the reaction. By repeating this process multiple times, usually 30, this will amplify the DNA exponentially.
· GE Illustra PuReTaq Ready to go PCR bead and tube
· Sterile nuclease-free deionized water (molecular grade)
· T-Streak plate with bacterial isolate
· Micropipettors and tips (P10, P100)
Adapted from &ldquoGE Illustra PuRe Taq Ready to go PCR beads&rdquo guide
- Obtain PCR bead tubes, which contain Taq polymerase (heat resistant enzyme) and other necessary reagents. Using a sharpie, label the top of the tubes with PCR reaction number assigned in class. Make sure not to accidentally rub this off when handling the tube and double check when you put the tube into the PCR machine that your labeling is still visible.
- Add 25 &muL of Master mix (contains molecular grade water + 16S rRNA primers) into the PCR bead tube. The bead will start to dissolve and slightly effervesce.
- As you dispense the Master mix, insert the micropipette tip into the mix so that you actually see the small volume go directly into the mix.
- Using a micropipette tip, carefully touch the colony on the streak plate. A small, visible dab of cells that barely fill the very end of the pipette tip will provide enough DNA template for the reaction.
- Dip pipette tip into reaction mix and gently swirl for 5-10 seconds to dislodge cells. Cap the tubes. Avoid forming bubbles.
- Transfer tubes to thermal cycler.
- Select appropriate program&dagger to start cycling (about 2 hours).
- Once cycling is complete, remove tubes and incubate on ice. Follow your instructor&rsquos instructions about storage, and follow up protocols to quality test the PCR products and prepare them for sequencing.
***Protocol adapted from &ldquopuRe Taq Ready-To-Go PCR Beads&rdquo guide*
5&rsquo &ndash AGA GTT TGA TCC TGG CTC AG &ndash 3&rsquo
5&rsquo &ndash ACG GCT ACC TTG TTA CGA CTT &ndash 3&rsquo
2. 94 o C 30 sec &ndash Denaturation step
3. 58 o C 30 sec - Annealing step
4. 72 o C 1 min 50 sec (1 min per kb of DNA template) &ndash Elongation step
6. 72 o C for 10min &ndash Final extension step
Tip 2: Choosing the optimal agarose gel concentration
Agarose concentration has a big impact on the quality of separation of your sample or ladder on a gel. The longer the DNA fragment being analyzed, the lower the agarose gel concentration needed (see Table 1 below).
Agarose concentration affects the range of efficient seperation
|Agarose concentration (%)||Range of efficient separation|
Figure 2.(A) Agarose concentration can affect resolution of DNA ladders. (B) Band resolution is improved with an agarose concentration properly selected for the range of fragments to be analyzed.
Selecting Markers and Ladders for Gel Electrophoresis
DNA and RNA size markers contain a mixture of DNA (or RNA) fragments of known length, making them suitable for estimating the fragment length of concurrently run samples. They stain well with ethidium bromide and other common nucleic acid stains for visualization after gel electrophoresis. For more information and background, please visit the Introduction to Markers and Ladders.
We offer a variety of markers for nucleic acid size determination of sample separated by various gel electrophoresis methods.
GEL ELECTROPHORESIS TECHNIQUE: STEPS AND APPLICATION
Gel electrophoresis is one type of electrophoresis technique, and its procedure shall be highlighted in this unit. The following materials and steps and steps are employed in gel electrophoresis technique:
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- Agarose gel is used for performing gel electrophoresis in the microbiology or molecular biology laboratory. It is noteworthy that the agar powder used for gel electrophoresis is different from the powdered agar used for the preparation of routine culture media plates for microbial cultivation. In gel electrophoresis, agarosegel powder is used to prepare the gel. The agarose gel is prepared by mixing a particular amount of agarose powder (e.g. 1.5 %) in a buffer solution or deionized water. Agarose gel could be made with varying concentrations of agarose ranging between 0.6 % – 3 % and this usually depends on the size of the nucleic acid fragments the researcher wishes to resolve or separate. Larger fragments of nucleic acids are separated or resolved better in a gel with a lower percentage of agarose while smaller nucleic acid fragments are separated better in a gel with a higher percentage of agarose. To prepare 1.5 % agarose gel for example, measure out 1.5 g of agarose powder and dissolve same in 100 ml buffer or deionized water in a conical flask. Stir the mixture properly to break up all clumps and heat the mixture by boiling at a particular temperature (e.g. 1 – 2 min) in a microwave oven until the solution becomes clear as water. A Bunsen burner flame could also be improvised for heating the agarose solution in places where microwave oven is unavailable. The agarose gel solution should be heated until a homogenous solution is formed. After heating, the homogenate gel should be allowed to cool to about 60 o C before pouring gel onto the gel casting apparatus or slab. Agarose, a white powder and the buffer solution are the two basic components of an agarose gel and both needed to be heated sufficiently to make the gel required to run the gel electrophoresis technique.
- A toothed comb (Figure 1) is used to form wells known as sample wells in the agarose gel. Thus, the toothed comb should be placed into the gel casting apparatus or tray prior to pouring of the gel so that the wells will be formed appropriately. The toothed comb is removed prior to the insertion of the DNA samples into the wells. The number of wells or holes formed is usually dependent on the number of samples or organisms to be analyzed and thus the type of toothed comb used in agarose gel experimentation varies. Samples for gel electrophoresis analysis are individually inoculated or dispensed into each of the toothed wells using micropipette (Figure 2). Multiple pipette tips (Figure 3) also exist for multiple analyses during molecular biology experimentation.
- Dispense the cooled homogenous solution into the gel casting apparatus or tray (Figure 4). The pouring should be done slowly, and all air bubbles formed during the pouring should be removed using a disposable pipette. Air bubbles could generally affect the shape of the wells if allowed to settle around the comb.
- The poured gel is allowed in the gel casting apparatus for some minutes (e.g. 20 mins) so that it will set or gel to form agarose gel slab. The gel casting apparatus gives the poured gel its characteristic horizontal shape required for agarose gel electrophoresis technique. Once cooled and gelled, the gel is now ready for agarose gel electrophoresis experimentation. It is then inserted into the electrophoretic matrix or chamber in which a buffered solution is also added to. In practice, the agarose gel slab is submerged in the buffered solution in the electrophoretic tank.
Pipette the individual samples into the sample wells created in the agarose gel by the comb (Figure 5). Ensure that the pipette tip is changed for each sample to be pipetted. A DNA fragment or ladder (with known or standard size) is added in one of the wells (usually the first well) and the ladder is used to compare the separated DNA fragments (with unknown sizes). In some agarose gel experimentation, ethidium bromide (EtBr) solution is added alongside the DNA solution to be analyzed. However, the EtBr is usually added to the prepared gel after cooling and before pouring onto the gel electrophoresis tank.
EtBr act as a chemical staining agent which helps to visualize the DNA bands or fragments after the electrophoresis experimentation. (EtBr is a dye that binds to DNA and clearly marks the position of the individual DNA fragments). In some agarose gel experimentation, the staining dye (in this case EtBr) is not added alongside the DNA solution to be electrophoresed. But it is added prior to or after the electrophoresis analysis since its main function is to aid the visualization of the DNA fragments. Note: EtBr is mutagenic or carcinogenic in nature, and thus should be handled with care. SYBR Green, a nucleic acid gel stain is another staining agent that could be used in gel electrophoresis technique to visualize separated nucleic acid fragments. However, EtBr solution is the most commonly used dye in gel electrophoresis experimentations and it is critical that the researcher wears gloves when handling EtBr since the dye is a mutagen and could easily be absorbed by the skin to cause health problems in the individual.Figure 5. Illustration of loading samples onto wells created on an agarose gel. When loading the samples, the pipette should be held at an angle in order to avoid puncturing the wells. Punctured wells will cause the DNA samples to leak out into the buffer and/or electrophoresis tank and this will cause contamination of the electrophoresis process.
- An electric current (e.g. 100 volts) is passed through the gel and the process is allowed to run for the appropriate time limit. DNA, a negatively charged molecule moves from the negatively charged electrode (cathode) towards the anode (positive electrode). The DNA moves through the gel matrix, smaller molecules move faster than the larger molecules. The electric charge or current is switched off once the electrophoresis process is completed.
- Separated DNA fragments is visualized under UV light and photographed after soaking the gel slab in EtBr or any other staining dye.
Alberts B, Bray D, Lewis J, Raff M, Roberts K and Watson J.D (2002). The molecular Biology of the Cell. Fourth edition. New York, Garland, USA.
Cooper G.M and Hausman R.E (2004). The cell: A Molecular Approach. Third edition. ASM Press.
Dale J (2003). Molecular genetics of bacteria. Jeremy W. Dale and Simon Park (4 th eds.). John Wiley & Sons Ltd, West Sussex, UK. Pp. 312-313.
Lewis R (2004). Human Genetics: Concepts and Applications. Sixth edition. McGraw Hill Publishers, USA.
Robert L. Nussbaum, Roderick R. McInnes and Huntington F. Willard (2001). Genetics in Medicine. Philadelphia, USA. Saunders publishers.
Sambrook, J., Russell, D.W. (2001). Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor Laboratory Press, New York.
Tamarin Robert H (2002). Principles of Genetics. Seventh edition. Tata McGraw-Hill Publishing Co Ltd, Delhi.
Twyman R.M (1998). Advanced Molecular Biology: A Concise Reference. Bios Scientiﬁc Publishers. Oxford, UK.
Agarose gel electrophoresis troubleshooting - (May/01/2009 )
1.if u're putting EtBR in the microwave-melted agarose in TBE/TAE..don't put it until the solution is cool!if u put EtBr when its boiling hot..it will degrade.
2.Try changing the loading dye?maybe it has too less glycerol?
I'm working on a new lab and i'm having a problem with my gel.
Why my DNA samples are running like this (see the figure)? The two lanes are 100 bp DNA ladder. All samples look the same. 1% Agarose gel, 80V 90 mA. I just don't have enough resolution with small fragments.
Brand new DNA ladder (promega)
Brand new agarose
1% agarose will not resolve 100 bp fragments well under any circumstances. Try 2% agarose, or (better) 3% Nusieve 3:1 agarose.
Did you run your gel long enough?
I use a 2.5% agarose and use a 100bp ladder. We use Seakem Agarose here and for better resolution, I would suggest using metaphor. Anyways, just increase the % of agarose to 2 or 3 % and you should be fine. Also, you should either stain with EtBr longer or add more EtBr when you make the gel. With little resolution and enough EtBr, the bands are blinding bright but yours are giving a weak signal.
Thank you guys, I tried a new agarose, and the resuld was really better!
I have a similar problem: I have nice bands of DNA and marker on top of the gel (the side where the wells are), but exactly at 1/2 of the gel, they disappear. So no bands there, at least the marker should be visible.
I use 0.5% TBE Buffer (pH about 7-8 according to pH paper), tried different loading dyes, and stain with texasred (tried 1/2 to 1.5 hours, with 10-20ul per 100ml buffer).
Any suggestions why this happens?
I used SYBR Green and nothing like that happens.
Ninana, the problem you are having with half of the gel unstained is caused by migration of the dye towards the negative electrode, while the DNA is migrating toward the positive electrode. This leaves the lower part of the gel unstained, since there is little dye left in that region. You can solve this problem by adding dye to the buffer tank in the positive electrode region, or by post-staining the gel.
thanks for the answer. But I do post stain the gel, so the dye is everywhere. but:
Does agarose go bad? - (Oct/05/2010 )
I've started work in a new lab for a month or so now but I've not been getting good resolution of bands >3kb in the DNA ladder. The lower bands are defined but those above 3kb are thick and smear. I was using TAE and tried 90V-120V. Higher voltage seemed to improve the resolution but still nothing as good as what I used to get in my last lab. Using new TAE didn't improve the resolution. I thought it could be the buffer, since I used SB in my last lab. However, I still don't get good resolution. I tried new DNA ladder, still no difference. I'm now wondering if the agarose is old (based on the batch no., it's 2007) and has degraded or changed in some ways (absorption of moisture?) detrimental to resolution. How long is the shelf life of agarose? Does old agarose give poorer resolution?
I do have one agarose, still using from 2007 and is still works fine.
Perhaps you should try 0.5X TBE for your gel and running buffer.
what concentration of agarose are you using? if the concentration is too high, you wont get a good resolution for the large bands, if too low no resolution for the smaller ones. usually 1xTAE with 80 V should produce good results for a minigel.
Have you tried to use less of the marker? Usually we use half the recommeded concentration and get perfect results (you just need to be aware of the dilution when doing quantifications).
I've already tried TAE and SB but both gave suboptimal resolution, so I'm suspecting something other than buffer. I'm using 0.8% agarose. It's on the low side so if any problem should arise, it should be resolving the smaller DNA but I have problem with the larger ones. I'm using 1/5 the recommended concentration of DNA ladder actually (100 instead of 500ug).
I've done these for almost 5 years at my last lab with no problem. Other changes are (1) using Gelstar here instead of ethidium bromide (2) casting gels in a mold instead of on a glass plate (yes, ONLY glass plate, relying solely on surface tension to form the gel thickness) (3) different power supply.
Are you using another brand of agarose than in your previous lab? there are huge differences here (also with the cheap agarose). No experience with Gelstar, but could be a reason. do you have the possibilty to try EtBr on your gel somewhere?
It's the same brand of agarose (Sigma, for routine use) but a smaller packaging. Too much hassle trying to use EtBr at my place (bureaucracy), so maybe I'll try post-staining and see if there's any difference.
is it the same lot that you used before. there may be lot to lot variation (personally, i think the difference may be due to storage conditions).
The gel chamber might also have an influence, some chambers are "better" than others (perhaps a design problem of some manufacturers). And of course the length of the chamber. The longer the migration distance, the better the separation.
Agarose: Only same brand, or also same type of Agarose? As written before, there are many different types with different characteristics (EEO values, strength, melting point, etc.)for different purposes.
I'm not sure if the current bottle is from the same batch, probably not since I'm in a different country now. Storage condition may differ. It's way more humid here, though air conditioned. Also, the current bottle was in the fridge when I found it whereas we used to store it at room temp at my last lab.
The gel chamber is a bit different by I think both are by Bio-rad. Size wise, they are similar if not identical. The agarose are the same type from the same company (Sigma, Agarose for routine use). Maybe I'll get some from the next lab and see if there's a difference. I think I'll get someone from my last lab to check the dimensions of the chamber for me too.
Is this a different brand of ladder than you used in your old lab? And have you tried a different brand of ladder in your gels? I recently bought a new, cheaper ladder and that's exactly what happened to me - and their technical support wouldn't respond to my emails!
Nucleic Acids and Oligonucleotides:
(Samples of DNAs of known size are typically generated by restriction enzyme digestion of a plasmid or bacteriophage DNA of known sequence).
The equipment and supplies necessary for conducting agarose gel electrophoresis are relatively simple and include:
- An electrophoresis chamber and power supply.
- Gel casting trays, which are available in a variety of sizes and composed of UV-transparent plastic.
- Sample combs, around which molten agarose is poured to form sample wells in the gel.
- Electrophoresis buffer, usually Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE).
- Loading buffer, which contains something dense (e.g. glycerol) to allow the sample to "fall" into the sample wells, and one or two tracking dyes, which migrate in the gel and allow visual monitoring or how far the electrophoresis has proceeded.
- Ethidium bromide, a fluorescent dye used for staining nucleic acids.
- Transilluminator (an ultraviolet light box), which is used to visualize ethidium bromide-stained DNA in gels.
NOTE: Always wear protective eyewear when observing DNA on a Transilluminator to prevent damage to the eyes from UV light.
For this we take 2ml of TAE stock solution in an Erlenmeyer flask and make the volume to 100ml by adding 98ml of distilled water. The 1x working solution is 40 mM Tris-acetate/1 mM EDTA
It is important to use the same batch of electrophoresis buffer in both the electrophoresis tank and the gel preparation.
For this usually 2 grams of agarose is added to 100ml of electrophoresis buffer.
Agarose Concentration in Gel (% [w/v])
Range of Separation of Linear DNA Molecules (kb)
What are some possible causes for DNA ladder smear?
I was analyzing my gel after electrophoresis yesterday and I noticed that not only my bands were smeared, the DNA ladder was also smeared as well. I understand the common causes of band smearing but what can cause the DNA ladder to smear?
Comb cleaning is something I find people overlook fairly regularly. Good recommendation!
Can it be due to dirty TAE running buffer? Because my lab only change the running buffer after 5-6 gel runs
This looks to me like an overheated gel or one cast with something less dilute than 1x TAE (water?).
Too much salt in sample too much salt in wells expired gel poorly poured gel overloaded gel.
Running too fast. That used to get me (but I was always impatient running gels).
My vote is overloaded gel in this instance. Dilute ladder 1:10 and try again.
You didn't let the gel set properly and ran it before it was solid. Leave it in the cold room/fridge for 10 mins or on the bench for 30 mins.
I agree, this or an issue with gel composition seems most likely.
Should not be the case. I left the gel to set for nearly an 1 hour before loading my samples
Did you make your gel in water by mistake? I've similar things happen when the agarose is dissolved in water instead of buffer.
Nope, pretty sure I used 1X TAE buffer
Did you figure this out? One of my labmates had the same problem and it was because she was adding way too much loading dye to her samples. Make sure that the final concentration of loading dye is 1x after adding it to your sample! :)
It looks to me like you ran it too fast, which caused it to get too hot and the gel melted while running. It could also be that your wells aren't deep enough, so the ladder&DNA are meandering on the top of the agar. The top is more likely to have imperfections from the pour and things will run wonky
Agarose gel Ladder smear - Biology
During this laboratory you will use agarose gel electrophoresis to separate DNA fragments which have been generated by digestion of your plasmid DNA with restriction endonucleases. You will then use a molecular size marker (a 1 kb ladder) to generate a standard curve of mobility vs. log bp and use the standard curve to estimate the size of the fragments separated on the gel (See below for a description of Gibco-BRL's 1 kb ladder and a picture with the sizes of the marker fragments)
1. Make 25 ml of a 1% (w/v) solution of agarose in TAE buffer.
2. Weigh the container with the mixture and record the mass.
3. Heat the mixture to boiling using the microwave oven. Examine the flask and continue boiling if any agarose is undissolved.
4. Weigh the container with the mixture again and add deionized water to compensate for loss of mass during boiling.
5. Allow the agarose to cool for 3-5 minutes at room temperature before pouring the gel.
6. Make sure the wedges are in place firmly against the ends of the casting tray. Pour all of the agarose solution into the casting tray, being careful not to overflow the tray. Add the comb and leave the gel to cool and solidify.
1. While the gel is cooling, prepare the DNA samples by adding 1 uL of tracking dye to 5 uL of each restriction digest. The tracking dye is bromphenol blue in a 50% glycerol solution. Adding tracking dye to the sample will increase its density so it falls into the well of the gel and provides a visible marker to monitor the progress of electrophoresis. Also prepare a molecular size standard by mixing 5 uL of the 1 kb ladder with 1 uL of tracking dye.
C. Loading and running the gel
1. Remove the wedges from the casting tray and fill the buffer reservoir with TAE buffer until the buffer is 1-2 mm deep over the gel.
2. Carefully remove the comb by lifting it straight out of the gel slowly.
3. Carefully pipette each mixture (6 uL) into a well in the gel . See the demonstration by the instructor before doing this step. Load one well with the prepared 1 kb ladder.
4. After all the lanes have been loaded, connect the leads from the power supply to the gel box. Make sure the gel is oriented correctly (wells at negative [black] end, DNA will "run to the red"). Set the output level to 100 volts and turn the power on.
5. Run the gel until the tracking dye is approximately 3/4 the way across the gel.
D. Staining the DNA in the gel with ethidium bromide .
1. After turning the power off, remove the gel from the gel box and submerge it in the ethidium bromide staining solution. WARNING: ETHIDIUM BROMIDE IS A POWERFUL MUTAGEN. USE GLOVES WHEN WORKING WITH IT. Allow the gel to stain for 5 minutes.
2. Remove the gel to a tray of water and allow it to destain for 1- 5 minutes.
1. Place the gel on the transilluminator. Place the clear plastic shield over the transilluminator window before turning on the UV light. WARNING: THE TRANSILLUMINATOR EMITS SHORT WAVE UV LIGHT WHICH WILL DAMAGE SKIN AND EYES, DURING PROLONGED EXPOSURE. BE SURE THAT PROPER SHIELDING IS IN PLACE BEFORE TURNING ON THE TRANSILLUMINATOR.
2. Turn on the transilluminator ( BE SURE THAT PROPER SHIELDING IS IN PLACE ) and look at the gel. Confirm the presence of DNA (orange bands) and turn off the transilluminator.
3. Remove the plexiglass shield and position the styrofoam shield and camera on the transilluminator. Turn on the transilluminator, wait a few seconds for the light to come on and shoot the picture by squeezing and quickly releasing the trigger on the camera.
4. Turn off the transilluminator. Remove the white tab from the film holder. Then pull out the photograph by grasping the black tab and pulling it completely out of the camera.
5. Wait 30-45 seconds and peel off the print from the backing. Don't touch the inside of the backing which has a photochemical gel on it. Dispose of the backing in the trash can.
Description of 1 Kb Ladder from Gibco BRL:
The 1 Kb DNA Ladder (U.S. Patent No. 4,403,036) is suitable for sizing linear double-stranded DNA
fragments from 500 bp to 12 kb. The bands of the ladder each contain from 1 to 12 repeats of a
1018-bp DNA fragment. In addition to these 12 bands, the ladder contains vector DNA fragments that
range from 75 to 1636 bp. The 1636-bp band contains 10% of the mass applied to the gel.
September 20, 1999 Copyright (C) 1996, Ivor Knight and Jonathan Monroe . All rights reserved.