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DNA Gel Electrophoresis - Science method

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I have loaded my qPCR products onto a 2% agarose gel and ran them at 60V for 1 hour. I ran 2.5ng of cDNA in a 10uL qPCR reaction. As you can see in my image there is smearing of all bands in the heavy direction. I have tried reducing the amount of sample loaded and switching to TBE buffer and this helped slightly but still gives the smearing that is shown in the attached image. What else can cause this?
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You can quantify your obtained DNA by checking the concentrations. Concentrations lower than 1.7 might not give the desired bands.
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Hi all, just a small question.
I run DNA gel using NEB 6x DNA loading dye as the loading dye and Gel red as DNA stain. Sometimes when I have very heavy DNA bands (e.g., after PCR) I can even see the red DNA bands (a bit faint though) directly in the agarose gel without using UV light. What could cause this? As far as I know, the loading dye serves to track the electrophoresis progress and precipitate the DNA sample into wells but has no ability to bind to DNA, while the DNA stain does help visualise DNA but is only visible under UV (at least Gel red requires it). So I am a bit puzzled here.
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Gelred has a second longer wavelength absorption of around 500nm as well as the stronger absorption maximum around 280nm and an emission spectrum at around 600nm. This makes the blue part of the visible spectrum in normal light conditions capable of exciting the gel red which then produces an emission also in the visible range
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Hi, just looking for some help with this. I can't for the life of me figure out what's causing this banding pattern. All my bands are producing this smily face pattern and neither I nor the rest of my research group can work out why this is happening to just my gels.
This is 1% agerose, run at 120V for 45 mins (it's a small gel).
Similar banding at 100V.
Tried using new buffer.
Loading 12 um of sample with 2 um of purple loading dye.
The agerose and buffer are microwaved for 2:20 (for 100 ml).
3.5 um of gel red is then added after the microwaved solution is allowed to cool for a few minutes, when is is mixed in by swirling it around a few times, before pouring the gel.
Any help with this would be much appreciated as these gels cannot be presented for obvious reasons.
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This effect often occurs when you add too much DNA in the wells. My suggestions would be, (1) add less DNA (about half of whatever you adding now) or wider well; (2) Voltage 80-90; (3) use fresh buffer
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I have made two controls and exposed them to the same conditions , but I found a problem in which that one is smeared and the other is not.
Condition : 20 min under UV radiation then incubation for 3 hr. in water bath 37c then I have loaded the sample .
Can UV make this change ?
Does UV has a random effect?
The controls contains cDNA
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UV light will nick dna strands and the damage is worse with shorter wavelength UV light and also with increasing time of irradiation. In addition incubation at 37 risks DNASE degradation of dna if the incubation medium is not sterile and this may have made the difference between your 2 samples
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Hi everyone,
I have a very troublesome time when I trying to build the construct of my gene. Since the gene sequence of my target insert is toxic due to the leaky expression, I used the competent cell that ordered from NEB company: NEB® 5-alpha F' Iq Competent E. coli (High Efficiency) to reduce this problem, and I encountered trouble when I tried to extract the DNA from the starters.
I used traditional digestion to get my insert and the vector, the I did ligation. After the colonies grew on the ampicillin plats, I did colony PCR by the use of my insert primers, it shows the very good result that my insert is inside of the gene of the colonies' cell.
Then I used the colonies to grow the starters (O.N. in liquid LB+amp.). I also did the starter PCR, but in the comparison of the positive control (the plasmid where I get my insert from), which has strong right size band; the samples shows nothing.
After that, I redid everything, and miniprep (i.e., the DNA extraction). In addition, I digest the extracted plasmid. The intact plasmid looks 2kb smaller compared to what it should be, also the digested bands.
What could be the problem during the process? If the idea that using low-copy number e. coli is not really working, is there any other way that I can use to build the construct with this toxic gene? My aim is to transfer the gene into tobacco and produce protein.
Thank you so much and looking forward to hearing any constructive suggestions.
Best
Ruojin Tian
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I have seen these positive results before from colony PCRs when you use the colony directly from the transformation plate. If you are using primers inside your insert and a good DNA polymerase, you can amplify your insert from the fragments of DNA that are spread over the plate, not inside the cells. This DNA comes from the ligation you used to transform the cells and the colonies that you obtain contain a empty vector.
To solve the problem with the colony PCR, you should use primers located in the vector that produce amplification of your fragment. If the fragment is really long, you can use a primer inside the insert and other in the vector.
Concerning the toxic expression of your insert, do you know if the expression is due to your vector or is your insert which is been recognized by the transcription machinery?
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From the standard text, I found that ethidium bromide intercalates the double stranded DNA, then how single stranded DNA can be visualized by ethidium bromide? I am curious about different visualization methods and mechanism?
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I agree with Mohsen Ashrafi . In addition , Ethidium Bromide is carcinogenic dye and there are another dye more safety like SYBER GREEN.
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Hi, I want to ask what is DGGE marker used for? Since there almost no commercial DGGE marker. I can make my own marker by myself, but does the marker make any sense when you analyze the DGGE gel ? 
Thanks!
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@Wolf T Pecher I am still not getting it right ..... I already optimised PCR with another master mix. I got a clearer band on AGE. But my DGGE still turned out to be aweful. I made a 6% gel with 30%-60% gradient. All buffer and reagents used are freshly prepared with molecular grade reagents too. What could I be doing wrongly?
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Hi everyone,
I perform a PCR + Restriction Digestion (single cut, same at both sides) over a metagenomic library. After that, I am trying to purifiy DNA fragments between 1-4kbp long from the smear I obtain after running the PCR in an agarose gel. Agarose gel is 0.8% and I run it at 100V 40 minutes. I carefully cut the band between 1-4kb and the yield or DNA recovery is quite good. The problem is that, after cloning it and taking a look at the size of the inserts, 90% of them are much smaller than 1kb.
What would be the best DNA Electrophoresis conditions (% agarose, Voltage, time ...) in order to maximize the separation between 1-4kb fragments from the smaller ones?
I believe that even a small quantity of small fragments after purification will be over represented after cloning protocol so, every tip in order to get rid of them as much as possible will be very welcome.
For tIn the ligation step I tried different ratios vector/insert (3:1 ,1:1 and 1:3) and temperatures (RT, 16 and 4ºC). The size distribution is basically the same.
Thanks a lo beforehand
Jorge
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Can your restriction enzyme bind to the DNA fragments after digestion? Check the information provided by the manufacturer. If that is the case, use DNA loading buffer containing SDS in order to disrupt the protein DNA-interaction so you make sure that bands that run between the markers 1kb and 4kb are actually fragments larger than 1kb.
Good luck
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How would I know exact pore size of agarose if i make X% of agarose? for example I made 4% agarose then what would be pore size of agarose gel?. Please give your valuable inputs. Thanking you!
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Hi all,
I just ran a gel electrophoresis that shows a bubble shape in the bands of the ladder. No other row had this shape, and I haven't seen it before. What causes this? Is there anything that can be done to fix that?
(I'm familiar with the crescent shape and have been making changes to fix that - lower voltage, lower sample volume, etc!)
Thank you for your help!
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examine the gel carefully.The usual reason for this effect is an air bubble in the matrix of the gel causing the dna to flow round it and so slowing just that bit of dna down compared with the rest of the band of dna
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I am planning to run some of the PCR products on a gel to identify the products of interest, perform a gel extraction, and do sequencing. My products of interest will be approximately around 300-550b in size. In order to get a band with a good resolution I have decided to use TBE instead of TAE and 2% of agarose. Do you think that 2% of agarose is too much or I should go for a low concentration (i.e 1.5%)? I'd also like to know the optimum voltage and time to separate my products (300-550b) in either 1.5% or 2% agarose gel. Please assist with the numbers.
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5-10 V/cm for DNA size <1 kb 
4 – 10 V/cm for DNA size between 1-12 kb 
1-3 V/cm for DNA greater than 12 kb.
further see the link
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Please find attached a photo of the problem; After a simple Agarose gel electrophoresis of DNA with EtBr i find the gel "divided" in two parts. Analizing at the gel at 259nm it seems that the first part nearer to the dwells is more whitish while the second part seems "black" as aspected.
I Hope that the question is sufficiently clear, ut looking at the ohoto you will have a more clear vision of the problem.
Any suggestion on which could be the problem( maybe light-scattering.....)?
Any suggestion on how to solve it?
thanks for any advice and the help will be very appreciated
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EtBr does move through the gel if you run the gel for a long time and if there is no etbr in the running buffer leading to a dark region at the bottom of the gel. You can still see your dna but if you think that you are missing any dna bands you can take the gel out and sit it in buffer with etbr for 20mins then the whole gel will have a brighter background but you are seeing your dna so what you are doing is working
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I performed agarose gel (1%) for PCR products of COX-2 from saliva samples of patients suffering from periodontitis. I could not see any bands and therefore in order to check if the RNA was isolated appropriately, I also performed agarose gel electrophoresis and again could not see even a band of RNA. Can anyone suggest where we are making mistake? 
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Revise the whole experiment from the first like sample collection and the final stage. Depend on other person is not good.
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Currently, i am thinking about GelAnalyzer software. Please help me with the analysis and band scoring. I have used 100 bp ladder.
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Size differences of 1-4 bp cannot be reliably assessed by agarose gel electrophoresis, due to the thickness of the bands. In your case, looking at both gel pictures, it seems that all samples contain fragments having the same length. The fastest migrating band may correspond to primers, if this is an analysis of PCR reactions
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I have obtained two bands from the DNA samples of Glaciozyma sp.
Has anyone works on Glaciozyma encountered the same issue.
the second band (~5000bp) is clear and distinct doesn't look like RNA contamination.
i have repeated the same extraction from a different batch of colonies cultured at different time (liquid culture)
and consistently they all have two bands.
this is a 1% agarose gel with 1kb ladder
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Hi Utsa, thanks for your recommendation. I will try to search it then.
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Dear all,
As part of an experiment I need to extract a DNA fragment ( from a restriction digest of a plasmid) out of a gel (0.8 %). I use LED light to visualize my band and cut it out. I use the QIAquick Gel Extraction Kit. I've done this a few times but every time the yield is low (<10 ng) and there is high contamination. I've made sure the agarose is completely dissolved, added an extra wash step, let the column stand longer with the elution buffer and even tried it with a new kit but there is no change in the outcome. Does anyone have any experience with this or know a solution?
Thank you in advance!
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Hi Paulien,
I use the same kit as you do for gel extraction, and I am able to extract DNA of about 8 kb with no problem. I always get good yield using the kit, so that shouldn't be the issue. Here's what I do:
1- I digest around 10 ug of my plasmid and load immediately upon completion into a 1.2% low-melt agarose gel. (5 ug should be okay too, but I would definitely suggest increasing the agarose concentration)
2- I run the gel at 130 V for about an hour. I've found that longer running time and a low voltage lead to substantial DNA diffusion out of the gel, resulting in very low yield.
3- To elute my DNA I use warm water (about 40-45 C) and let it sit on the column for 10 minutes before I centrifuge it.
This method has always worked for me, I hope it does for you as well! Good luck!
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When I run a gel on the extracted DNA from my samples I always notice smear with varying degree of strength what do they mean?
a picture is attached to clear out my point
I store the samples at-20 for 4 months before beginning my DNA extraction can this storage condition cause degradation of DNA ?
I used 0.7% agarose gel and run on 200-220 volt for 20 min
I used iagen blood and tissue spin-column protocol
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Greetings,
The DNA degradation might be due to the nuclease released during homogenization. Might I suggest preparing 1mL 1X PBS + 2uL EDTA, and proceed with the kit's protocol. During homogenization, do perform it in an ice box to lower the nuclease activity.
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Whether I should use Scion image or Imaage J software for DNA bands analysis?
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image j- image j2 fiji, gelquant,
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Hi
Is there a real correlation between % of cell death and the efficiency of DNA fragmentation assay?
I want to perform DNA laddering from suspension cell sample with low % of dead cells (less than 30%) . Could it work successfully? or should I take sample with more than 70% dead cells to see clear DNA fragments? Is there a conventional threshold in this case?
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I recently did chromatin extraction from brain nuclei samples. Since the individual sample amount is too small, I pooled 10 samples together (~40 mg). When I used the Epigentek Chromatin Extraction Kit, it is suggested on their protocol to shear at 2*20s and there was detectable dsDNA concentration right after chromatin extraction. Following their instructions for reverse cross-linking to get input DNA (pre-mixed solution from the kit with 2.5 uL proteinase K, 15 min incubation at 65 C then 10 min at 95 C), there was no band showing up and the dsDNA concentration after this input DNA extraction was also very low (~10 ng/uL on average and the loading amount on the gel was ~80 ng). I then tried with the homemade recipe for chromatin extraction (freshly made 1% formaldehyde cross-linking for 10 min rotation at RT, then quenched with 0.125M glycine for 5 min rotation at RT; lysis [0.01M pH8.0 Tris-HCl, 0.01M NaCl, 0.2% NP-40 with protease inhibitor cocktail] using needle (referred to the protocol from ThermoFisher website); extraction [1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS and 0.004% sodium azide in 1x PBS] and sonicate for 10 min (30s on, 30s off on ice). The input DNA was then reversed cross-linked with 6 uL 5M NaCl, 2 uL RNase A and 2 uL proteinase K at 65 C for 4-6 h, phenol-chloroform-isoamyl extracted with ethanol precipitation at -20 overnight and eluted in water. There was no detectable dsDNA right after chromatin extraction but the concentration for input DNA was over 1000 ng/uL but the ratios were only about 1.4 as when using the kit. Using about 120 ng DNA on a 1.2% gel, there was no band at all. I am very confused by these results. Have any of you confronted with such scenario before? Any suggestions? Thank you!
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Yang Xiao, I hope you have found some solution for your problem and you were able to rectify it. For cross-linking, people have tried some other cross-linker, but always with formaldehyde. I am not sure how it helps but you can give it a try. You can find some answers for that here: https://www.researchgate.net/post/Does_anybody_know_of_an_alternative_to_formaldehyde_formaline_in_Chip-Seq_experiments
For the quantification purpose, if you don't have Qubit, you can also simply do a PCR to check if there is any DNA in your preparation. If you want to quantify the amount, you can do qPCR which will give you a fair idea of DNA amount. You can check the website of the product that you use for qPCR for more details on this. I am sure all of them have some guidelines. However, it does not tell anything about the quality of your sample. So, I will also check the samples using Nanodrop for quality.
Hope it helps.
Best,
Vikas
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I was wondering if anyone knows why this may of occurred? I have not had it happen before. I need to write a lab report for these results, so as much detail as possible would be greatly appreciated.
The method I used is below:
Agarose gels (2% (w/v)) will be used for this lab and the gel will run for approximately 30 minutes at 100 volts
There are 14 wells for each group, this is more than enough for you to load all PCR products and have at least one lane for a ladder, you do not have to load each well on the gel.
To load the gel add five microlitres of your sample and one microlitre of loading dye. The loading dye is Cresol Red (0.25% (w/v)) and therefore different to what you have used previously.
On an agarose gel, Cresol Red runs at approx. 100bp therefore is a more suitable indicator to ensure your samples don’t run of the end of the gel/into the samples below.
Add five microlitres of DNA ladder (100bp ladder).
Thank you!
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Your wells formed badly during preparation of gel thus you have v shape of bands.
Most probably reason of smear is your buffer is old and use over and over.
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Can you use e-gel ladders on normal 2% agarose gels?
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yes, they work well.
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I need a huge concentration of restricted product. So can i just digest them in a 50 uL reaction with high volume of enzyme (say 5-7uL). This is just to run the digested product in a single gel and do a single shot gel elution.
Thanks in advance
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You can always increase the concentration of your desired digested product using a speed-vac or putting the tubes in a warm heat block with the lid open. Out of curiosity, why does the concentration need to be so high?
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I am a gene expression guy, but my current project has pulled me kicking and screaming into the protein realm.  To that end I am preparing an expression plasmid for protein production in E Coli.  I have performed a double digest of the plasmid backbone, and the results were what I expected with the exception of a third band around 3.5kb in both digested and undigested samples.  I attach an annotated image of the gel.
Could this be either ssDNA or chromosomal DNA, both of which would indicate some issues with my midiprep?
The good news is the major product is the linearized plasmid I expect, but I would like to understand this other band for future reference.
I would greatly appreciate input from those of you who do this for a living regarding what this band is.  Thank you!
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It is not single stranded circles! It is partly rerormed/partly random looped cccDNA that resists restriction enzyme digest and can account for up to 50% of some DNA preps by alkaline lysis. This is a well-known problem and can be solved readily:
Sayers et al (1996) "Identification and Eradication of a Denatured DNA Isolated during Alkaline-Based Plasmid Purification Procedures", Anal Bioch, 241:186-189.
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I want to detect 15 bp of difference in DNA fragments of 350-385 bp length. Which method will be most useful? Agarose, PAGE or primer labeling and genotyping?
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Hey guys, just resolved a 14bp difference on a 3% agarose gel prepared and ran in TAE buffer, please see the attached picture.
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Hi everyone,
Yesterday, I run electrophoresis of PCR products, its length would be about 149 bp. Everything was ok but when I saw the result, I found a strange phenomenon. The lane of marker was normal but I did not know the reason why I got no desired bands. Especially, I noticed that on the top of the gel, there were several small bands.
Could anyone suggests about what its going on here?
Thank you for your time.
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Probably genomic DNA contamination
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I start with impure PCR products of >500ng/ul, 260/280=1, but peaks present at 260, and end up with peaks near 230, about 10ng/ul concentrations of 260/280=1.5 or so, which I assume is not DNA since there is no peak at 260. Is this due to contamination by guanidinium salts from the buffers? Or ethanol? What can I do? Gel electrophoresis with a 2% agarose gel for 25 min at 70V showed DNA of the desired sizes, though not necessarily large amounts, and after purification, there was pretty much nothing.
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I mainly purify PCR products to either clone into vectors or Sanger sequence. For these applications, the contamination from the Qiagen gel extraction kit has never been a problem.
For purifying DNA in NGS workflows, we use Ampure magnetic beads, so you could try that if the contamination from the gel extraction kit inhibits your second PCR, but I suspect it won't be an issue.
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I would like to know from your experience where the mistake is
Experimental steps include the use of sodium salt calf thumus and was diluted in TE buffer size 50 ml  and at a concentration of 2 mg / ml l . After dissolving DNA for 24 hours at 4 ° C was exposed to eletctron beam 6 Mev from elekta preise linac (dose = 80 Gy and dose rate = 5 G / min, field size 20 * 20)
 Then 50 mL DNA  was divided into two glass petri dishes and a depth of each DNA solution in petri dish was ~ 1.6 cm and 1.5 cm water equivalent material was placed directly above the two plates without placing the cap petri dish. Gel eletrophorsis were then performed after a week of exposure and the sample in that period was reserved for the refrigerator at 4 ° C
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do you expect to see visible degradation?. If one of those samples is a control then it is already quite degraded so any degradation of other samples will simply move already degraded dna a little lower down the gel but looking very like the control. Ionising radiation will cause single stranded nicks but dna is very long and double stranded nicking causing breakages will be quite rare. I think that you need a more sensitive technique than agarose gel to measure degradation. Possibly some of the bright signal in the well might be very large dna but could also be protein like histones.
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I would like to draw a DNA structure (both single strand and double strand).
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Hi,
"Avogadro" is a software that you can easily download and build your DNA and then save in PDB format
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So I just found a few boxes of unused E-Gels ( 4% High Resolution, Invitrogen™ /ThermoFisher) with an expiration date of 10/2017 in my lab. I'm wondering if anyone has worked with expired E-Gels before and they still function properly? I hate to have to trash them as that's quite a lot of money... Thanks!
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I have an experience of using commercial precast gels (2%) after date expiration. It was not from Thermo Fisher, but I think the same principle. In the case of intact individual pack it could be ok, but you shoud verify the hydratation and consistency (visual and/or by touching) . The gel may dry up, then the results of electrophoresis will be unsuitable.
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Need your suggestions...
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it is hard without a size standard but I agree with Geoff that if these bands are about 1kb and 1.5kb they are probably 23S and 16S rRNA and yes I do expect old lysed bloods to have degraded rna and indeed many of your samples show signs of degradation so I expect the samples with clean bands were the least lysed during storage ( probably those stored for the least time.DNA also degrades so often samples stored for that long before processing can be stored frozen with less degradation
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I have a procedure involves the using of polyacrylamide, but I don't have it! Can I use MetaPhor instead? What limits the using of MetaPhor instead of polyacrylamide?
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Thanks Paul for all these details...
Is MetaPhor agarose applicable in MSAP technique?
Regards.
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i had restriction digestion result and i would present it this week is there and advises academically
cheers
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if you posted a picture it would be easier to comment on the result
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I am studying the release of DNA from nanoparticles in acidic medium at different times (lanes 4,5,6,7,8 from the left), but no bands appear in the gel in contrary to the main DNA appeared in (lane 2). Different concentrations of agarose, and different initial loaded volumes of the nanoprticles were tried for observing the bands, but in vain. However, the gel documentation system can analyze these bands, and give the amounts and molecular weights of the separated fragments. What is the problem with the gel?
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Dear amir
Although i still do not know your aim of the study, but definitely there is good improvement in your DNA gel yield . the presence of two bands may reflects the possibility of DNA  melting or you may called it relaxation.the presence of high salts concentration make a shield to negatively charged phosphate groups in the DNA and affects as well their mobility,so, if you adjust the salt concentrations in the samples presented in lane 4-8 to be similar to those in lane 2-3 probably you can get similar pattern. May be you need to do one of the following:
first if you have a minicolumn kit for DNA extraction so you can use the column and apply your sample to it and do centrifugation to get rid of salts and the DNA will be adsorbed to the column resin . Then use the washing solution of the kit to do 3-5 washes of the column to get rid of extra salts and then use the 25-50 microliters of the eluting solution of the kit after drying the column by centrifugation and use the eluted DNA solution for a new electrophoretic separation and observe the effect.
Or you can apply your DNA sample on a small column packed with sepadex G-25 to desalt it and them apply it on gel. I hope these suggestions will help you.
. Good luck
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I used 0.8% agarose gel for PCR products and 1-1.2% gel for genomic DNA made with 1x TE buffer. But, I am confused that % increase in gel concentration  decreases the pore size and fragments to be moved  forward and if so, I would have to use 1.2% gel for PCR products and 0.8% for genomic DNA. Anybody please suggest me the concentration and voltage requirement for those both.
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chromosomal DNA =5V/ cm -1% agarose-1-2 hr
pcr product= 7V/cm-1.2 -2%agarose  (depend on product size)- 1.5-2 hr
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Hello, 
I have problem with loading wells in polyacrylamide gel. It looks like the gel around loading wells polimerize poorly. There is a lot of very thin layer gel in the wells after removing the comb. I have tried to flush it with the buffer, but the effect is way from desired. Any tips? 
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Hello Rafal 
1-You should increase for both "resolving and Stack"  APS and TEMED concentration just like 2ul
2- Mix it very well then pour it  
3- After pouring the resolving ..  You should incubate it at  temperature 37 for 20 minutes  Then pour the stack and also incubate at 37 until polymeriz . 
4 - Remove your Combs after adding running buffer
All the best 
Sally
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Hi all,
I want to seperate 4 DNA fragment (210, 175, 169, 41 bp). It is not neccessary to seperate 175 from 169 fragment. I could recognize the polymorphic variant without this.  I have tried with 0,7 grams of agarose and 35 ml of 0,5x TBE buffer, but the resolution was bad. What should I change? Maybe 3% agarose gel? Should i lower the voltage, I tried with 130V. I will be grateful for any help you can provide.
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I would suggest reading Brody et al. 2004 Biotechniques 37: 598-60. The suggestion some have made to run the gel at a lower voltage to minimize thermal diffusion of the small bands is only relevant if you use the usual (and not very good) electrophoresis buffers (TBE and TAE). What you really want is to run the gel as FAST as possible without the heating, and you just cannot do that with those buffers. They are really terrible for electrophoresis, because they are too salty (thus the thermal heating) and the contain Tris, which makes the problem worse, because Tris breaks down during the run and makes the conductivity problem and heating worse in a positive feedback loop.
By simply switching buffers, you can run very fast gels (say, 1000V or 100 V/cm). As Brody et al show, very small fragments (24, 40, 60 80, 100 bp), normally requiring polyacrylamide, could be resolved in1 mM lithium boric acid (or 1 mM sodium boric acid) in a 3% agarose gel in about 5 minutes (100 V/cm).  Make sure you use a good quality agarose with a low EEO value. You will also need to make sure you have reasonably low salt in your sample plus load buffer/dye. If your sample+loading dye is very salty, DNA migration will be retarded until the salts diffuse away.
Brody et al also have recommendations for the best simple salts and concentrations for separating other DNA size ranges (and RNA). But basically, either lithium (or sodium) boric acid in concentrations ranging from 1 mM to 10 mM will work for almost all your electrophoresis needs. You will save a lot of time on electrophoresis and on buffer preparation, as well as money, because the solutions are simple, low concentration, and cheap. You can buy the concentrated electrophoresis media and loading buffer pre-made (http://www.fasterbettermedia.com/) or you can make it yourself. (Note: I have do not have any financial stake in this company)
For an enlightening history of electrophoresis media see also Brody and Kern 2004 Anal. Biochem 333: 1-13. It is fascinating to me that TAE and TBE have remained so common when they are so poor at the job they are supposed to do. An illustration of the power of tradition and the inertia that can take hold in our routine work.
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I have run a few electrophoretic mobility assay gels recently using Hoechst-33258 and ethidium bromide in order to observe the changes in the mobility of the DNA when each are used in comparison to a blank sample. In the case of EB, the mobility of the DNA is reduced due to the extension of the DNA helix on intercalation, which makes sense to me, but when I run H-33258, I see an enhancement of the mobility of the DNA, which is what I don't understand. Is this observation a common occurence?
I'm hopefully going to use these gels as diagnostic tools for studying the interactions of a novel compound with DNA so I'd just like to make sure I understand what is actually happening with my standards
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Please refer to these references. It might be helpful.
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I am running a native gel to image DNA bands. Some of them are dsDNA and some are ssDNA. Used 10% PAGE. Loaded 50uL in each well with concentration 1uM (either ssDNA or dsDNA). Used 1x TAE Mg+2 as a buffer. Run the gel for 2.5h on 170v. Soaked the gel in EtBr for 30min, destain for 30min. Then image it under UV.
Any tips to make the ssDNA to show up?
Thanks in advance!
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Hi Abeer,
You're correct, the ssDNA that I've worked with are way bigger than yours. Sorry! :)
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Hello,
I'm new to practically everything involved with PCR, electrophoresis and EtBr gel staining. I am having trouble with visualising the gel using EtBr after electrophoresis; there is such an enormous background interference that I can no longer see the DNA-bands, given that they are rather subtle already.
I have tried removing excess EtBr after submerging using a paper towel but so far no results. Maybe I should mention that I don't wash the gel after EtBr-staining.
If anyone would know what to do, that would be greatly appreciated!
Thank you beforehand
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Use less EtBr, before casting the gels. Also, it is better to decrease the thickness of your gel. It effectively increase the contrast and quality of your figures. 
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I am running a 27kb long fragment of DNA on gel electrophoresis. I am using 0.5% agarose concentration and a voltage of 5 volts per cm of gel used. TAE buffer is being used. How long should I run my gel for to get the best resolution possible?
Thanks!!
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Hi, Florencia
I hope this will be helpful.
Most agarose gels:
1. 1% gels are common for many applications.
2. 0.7% good separation or resolution of large 5–10kb DNA fragments.
3.  2% good resolution for small 0.2–1 kb fragments.
4.  Up to 3% can be used for separating very tiny fragments but a vertical polyacrylamide gel is more appropriate in this case.
 TAE has the lowest buffering capacity but provides the best resolution for larger DNA. This means a lower voltage and more time, but a better product.
 Voltage plays important role in this case. I manage to get a good resolution at 1.5% agarose and running slowly at 80V in a very thin gel (>4mm), use cool (10-15 degree Celsius) 1% TBE Buffer and its gives good resolution. I think you can get to a resolution of ~50 bp or even less, but you'll need to have at least 1.5-2% gel (the higher the concentration, the better resolution, but the DNA runs slower), and run it for a long time. Varying the agarose concentration is going to be your best way for getting optimal resolution. (Higher concentration = smaller pore size = better separation). The recommended thickness for agarose gel is 3–4 mm; a gel thicker than 5mm will result in fuzzy bands and higher staining background. Similarly, the amount of running buffer to cover over the gel in an electrophoresis apparatus is 3–5 mm. Too much buffer will decrease DNA mobility and cause band distortion. If the voltage is too low, then the mobility is reduced and band broadening will occur due to diffusion. If the voltage is too high, the band resolution is reduced, mainly because of gel overheating.
A higher agarose percentage enhances resolution of smaller bands; conversely, a lower agarose percentage gives better resolution and separation of higher molecular-weight bands. If the wrong percentage is used, it can be difficult to visualize the DNA bands reliably.
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Hi everyone,
I ran some DNAs and saw that they looked normal on an agarose gel but very smeared on a polyarylamide gel. They smeared towards high molecualar weight band. I tried 0.5X or 0.25X TBE as running buffer but saw same result. Could there be an issue with my water?
Thanks.
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Most likely the problem is with your loading.  Unlike in an SDS-PAGE gel there is very little stacking effect in a TBE-PAGE gel and so if the DNA solution is not loaded very carefully into the bottom of the well or if there are irregularities in the shape of the wells then these irregularities will show up as distorted bands.  After pouring the gel and pulling the comb immediately wash the wells with a little 0.25X TBE to ensure any slight bits of unpolymerized acrylamide are cleaned out and examine the wells to ensure the they are uniform without defects. I can see from the spread-out shape of the bands that your wells were not completely flat-bottomed and had a slope back-to-front, hence when looking through the gel thickness the bands seems smeared or doubled as in the ladder. I also agree that you should try the above suggestion of loading less DNA, in the agarose gel there is some streaking of DNA in front and behind the major bands, acrylamide gels are much more sensitive so you are probably just picking up some more of this material.  Also, as a final point, it is important in my experience when running TBE gels to assemble the gel apparatus and pre-run the gel for about 5 - 10 min before loading any DNA in it. This ensures that any slight difference in the concentration of salts between the gel and the running buffer is run into the gel before the DNA is loaded and thus won't cause irregular migration of the bands.  
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I digested my PCR product with TspR1, and the sequence was cut into 3. Using restriction mapper, I was able to confirm the length of each fragment. When ran on gel, the fragment that was meant to be 70bp appeared at a point OVER 100bp
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Thanks to everyone. And yes Saha you are right with the procedure. the sizes of the cut dna is 131,70,23. The shift for 70bp was most obvious.
But I have been able to realise that one major reason is bcos of the enzyme and the way it cuts, leaving sticky ends.
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Hello, there is something that keeps bother my mind and my project. I ran a 25 bp ladder in 2% of agarose gel in 78V for 2-3 hours. The problem is, no matter how long you run the gel, still, only 1 band that would appear. I have tried diluting the stock in 1:9ddh2o and using the stock directly but the result would still be the same. The ladder was purchased about more than 6 months ago, maybe about a year and was not opened yet, until yesterday. I've tried 1,3, 5ul but there was only 1 band...
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If by a 25bp ladder you mean a ladder with bands going up from 25bp in 25bp steps, then what is the top molecular weight supposed to be? If it were still small (say <200bp) then you may have run the ladder right off the bottom of the gel.  But what I don't understand is, in that case, what is the single band.  I think it is most likely that what was in the tube you loaded was not what you thought is was, as it appears that you have a single and quite clean DNA band in each lane, running with you top dye marker.  Check the label on your 25bp ladder tube, and if you are convinced it is labelled as a ladder, ring up the manufacturers, quote the batch number and ask if they have had any problems. (In general,  when dealing with commercial products when you have a problem, ask the manufacturer if they have seen the problem before you do much else).
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I have 3 animal /group for control (C) and treatment (T) and used housekeeping gene (IC) for each. Now after running gel, Image j densiometric results - lets say are C - 11.9, 13.3, 4.4 ; T- 14.5, 11.4, 5.2 and IC for c -9.2,5.3,5.5 ; IC for t- 5.1,7.6,10.1 respectively. How to calculate 1. Expression level of T compared to C. 2. How to calculate fold change 3. Which type of t-test to verify significance.
Please Help.
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Nikhil: In today's standard, real time qPCR is a routine for fold changes, and densitometric data is less reliable and acceptable by many journals.  The reason: the linear range in a PCR reaction is often between cycles 12 to 32, and each pair of primers needs to be optimized experimentally before obtaining the data for analysis.  So, reviewers will definitely ask for that.
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I have also run the internal control gene. How to normalise, plot graph and in which units to express the data.
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  • You can normalize by dividing the band intensity of gene of interest by band intensity of internal control gene.
  • The ratio value of the intensity can be plot directly to the graph.
  • It will be express as relative quantification to the internal control gene.
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I am extremely confused PLEASE HELP. I watched two videos but I still do not have any clue as to how to interpret my results.
Attached are images of the gel electrophoresis. Samples are in this order: DS, ZZ (This one is my sample), PV, BS, RZ, AS, JN, AV, MW, K, J, IG, JM, AK, AW, C, AW, CS.
Image Cdk3: contains TAS2R38 (didn't come out so well) and CDK3 (DS and ZZ (mine) samples are to the left of the ladder)
Image Adhl: contains remainder of cdk3 samples and both ADLH and ADH samples together from each individual (tubes 3 and 4 consecutively) (JM did not have sample)
Image D1s80: contains remainder of ADLH and ADH samples and D1S80 to the right of the ladders. (C did not have sample)
Ladder used in the electrophoresis.
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dear Zaneta;
Cannot interpret your results since there are not expected pcr product size on your amplified genes. but we can tell about what and how are your produced bands in the gel.
on the first gel image top line left handed you had 8 samples as we see 4 of them showed band and 3 of them were showed the primer dimer (it may because high temperature of annealing, lots of cycles, or not extracted DNA samples well and my your primers do not work as well)  
also about the samples were showed the bands you have to know the size of your genes you interested if the expected equal to observed size you get correct results, if not you should change the anealling temperature by useing 3 degree temperature above and 3 bellow of your previous temperature.
on the right handed first image you had 4 samples no one showed tha bands even the second one next to the primer showed the broken fragments due to the high anealing temp. or lots of cycles try to reduce number of cycles and use defferent temperature. we had same results with one of the M.Sc student here we used 25 cycle and defferent temp. were solved the results.
and the belowe lines and the other images you have the same problems try to do some of these suggestion if you would like to:
1. if you have primer dimer try to reduce the amount of primers you used and if you use a few of DNA samples increase it ( about 30ul for RAPD PCR and above 50-100 ul for RFLP and specific primers). 
2. if you have more than one bands, reduce the number of cycles.
3. if you have more than one bands but some of them above your target bands and others below your target bands, use defferent teperatures. used touch down pcr.
4. if you have the dimer from the top to the end of the gel try to descover if any contamination, bad handeled materials specialy storage of DNA is not well. you may have broken fragments.
i am sorry if i am not servce you well
good luck
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I am working on methylation analysis of DNA (from blood/PBMC's). The technique which I will carry out is COBRA which is done by bisulfite conversion, followed by PCR amplification. Then based on the presence or absence of RE site restriction enzyme they make the cut. PAGE is carried out after that and then via densitometric analysis we can calculate the % of methylation.
However, few articles mention electroblotting as the technique of their choice instead of PAGE. Can either of you help me and let me know if I can do PAGE and still get the % of methylation instead of blotting.
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Hello,
Thank you so much! I will for sure check out the software. Is it freely available?
Also, as you are aware I am looking at RE digestion of the Bisulfite converted DNA. Would you like to suggest some apt RE for the same?
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in the gel, DNA ladder is not resolving into bands but is seen as smear. i checked the pH of the TAE buffer and made fresh buffer but the result is same. kindly help.
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Dear Manvendra,
Let me Know two things 
1. What base pair ladder u have loaded in Gell...
2. If your gel is 1% gel or 2% gell...
Because its generally seen that 100bp (Low base pair ) ladder can not be Resolve clearly and seems to be smear in appearance in 1% GEL.
Moreover multi time use of the same Gell by remelting also gives the u said results.
If i am not wrong your are trying to load 100bp lader in 1% agarose GELL.
If it so i request you to please prepare fresh gel and Load and Hope you will get and end solution.
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I have a question that may sound silly. Can meniral water (and by that I mean commercial water purchased with the purpose of drinking) instead of distilled water that is usually used for the preparation of agaorose gel and TAE buffer.
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Hi there,
For instance TAE is meant to contain only tris, acetate, EDTA and Na+. If preparing with mineral water the composition will be different (depending on what minerals you have in the water...) and the ionic strength will be higher therefore it will affect its properties for running agarose gels.
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This is my electrophoresis result of DNA plasmid preps on 1% agarose gel. DNA plasmid's size is 7037 bp. Lane 1 is DNA ladder 1kb, lane 2 and 3 are my samples. I think that the last band at 5kb is the supercoiled form of plasmid. However, the above strong band, is it the open circle form of plasmid? I think that it might be contaminated chromosomal DNA.
Is it right? Please help me to explain this result!
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I think its not contamination, its your plasmid. When plasmid DNA is isolated and run on an agarose gel, you may observe 2, 3 or even 4 or more bands. the first band is circular or single strand band. the second band is super-coiled  the third band is linear and. the fourth band is nicked plasmid. if you want to be sure you can use digestion enzyme to cut your plasmid or send your plasmid for sequencing.
Sincerely
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Hi everyone
I have read a paper recently as following
In this paper, it says "There is nearly no cross reaction among closely-related miRNA family members, which points to the high specificity of the assays"
"The percentage of cross reaction values was calculated between assay-specific and unspecific miRNA targets"
, But I am wondering is "percentage of cross reaction values" have any different to 2(ΔCt) ( ΔCt= Ct of unspecific -Ct of specific)? this is my first question
second is how  "percentage of cross reaction values"  count? I have search the internet for answer but can't found.
thx you all
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My best guess is that they mean fx that a20 fM synthetic template for le7a miRNA  gives Cq 25 in a let7a qPCR assay. Then when the let7c 20fM synthetic template is used in the same assay, it gives Cq 35, so  the difference (2^10= 1024) is calculated into percentages (So the Let-7a assay has ca 0.097% (1/1024*100) cross reactivity against let-7c targets at the given conc).
this is as I said best guess..........
best
thorarinn
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Do you guys know a cheaper alternative to sybersafe for running a gel?
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Ethidium bromide
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I haven't heard of the company, despite being based in Cambridge (UK) for many years. Thermo are starting to distribute these machines, but I haven't found any reviews on line.
Any help you can offer would be gratefully received.
Thank you in advance. Leeanne
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I use the darkroom UVITEC set to chemiluminescence only when I will reveal western blott membrane , I have never used or saw using this set equipment in this way to reveal DNA gel. Are you sure about that?
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Hi all, does anybody have a clue what's happening with my DGGE gels? My first 3 gels worked fine, but since then they have just been complete disasters. I've attached some photos of my gels.
I've tried looking up other Qs and As but the answer to all smearing problems seems to be "bad PCR", but the failed gels contained the same PCR products as the gels that did work.
Thanks so much for any input!
Lindsay
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Your problem might be a matter of using the wrong cleaning agent for cleaning your DGGE device and the GLASS PLATES.
When we first realized our problem we started to work on the PCR/ the gel/ the buffers/running conditions .... EVERYTHING ... however, it took us month to realize that the soap for cleaning our DGGE device caused such smear effects!
We had changed it without puztting much attention to it. In the new soap - there where some oily substances added that interfered somehow with the glass surface. We used new glass plates and switched back to our old cleaning agent (kind of scrub cleaner without any special ingredients). Ask the supplier of your DGGE device for the best cleaning agent to use!
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Looking for the Answer in details. 
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A good question. Please read this article at below link:
"Why aren’t SDS-PAGE gels horizontal?"
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Hi,
I am trying to get plasmid profiles from clinical isolates. I extracted them and visualized them in the gel electrophoresis. I tried to cut them with a 6bp enzyme XbaI to have more ideas on the size of the plasmids (anyway, I chose an enzyme randomly, since I don't know the sequence of the plasmid). I have some difficulties in the interpretation of the data and I attached the file (sorry the image is not great). They are 6 isolates and for each I run the cut and the uncut plasmid. I don't know how to determine the size of the plasmid and how to determine if I have more plasmids in my extract.. For example, as to the first isolate (K1), the cut  produces 2 bands, one of which is larger than the only one band visible in the uncut product.. in your opinion, could the bands be the two pieces after cut of the plasmid? If yes, why are they larger than the single band visible for the uncut plasmid? does it mean that the uncut product is a supercoiled form e migrate faster?...the other example, the cut of the plasmid extract of K5 produces different bands, again,  larger than the K5 uncut. Are they pieces of the same plasmid after cut or different linearized plasmids?Again, the fact that I have bands larger for the cut plasmid DNA can suggest that the uncut plasmid is in the supercoiled form? I used the 1kb ladder
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I agree with David's answer which is a better answer to the question you posed.
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I am trying to amplify a large DNA fragment (about 5 Kb) by PCR. I used a FX-PCR master mix (for long and fidelity PCR). The PCR condition is as follow: 95C 5 min, 35 cycles of ; 95C 30 sec, 53C 30 sec, 72C 5min. But after electrophoresis of my PCR product I have smear on the gel. I have tried gradient PCR and also changed the primer but still I did not get any band.
Thank you
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the important thing in long pcr is that the template does not re anneal to itself before the extension happens so all long pcr should have primers that anneal at very high temperature to minimise the time that the template has to become double stranded again. I would design primers annealing close to 72c and do the pcr in the presence of 1Molar betaine which keeps the template melted for longer by destroying the triple hydrogen bonding of C-G bonds so makes the template melt easier. A profile of 95c,70c,72c would work much better . 53c is very low for long pcrs
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I am trying to add Rf lines to pictures of my gels that have some minor distortions to aid in calculating MW for each of my fragments. I am a bit confused about the proper way to add and move the Rf lines, though, to make the calculations more accurate. 1) In the attached image, my wells are slightly slanted down to the right. Would I add the first Rf line just below the wells, and edit it so that it slants down? 2) Do I need an Rf line for each of my ladder bands? And would I also edit this so it is slanting down to the right? The program does not automatically create Rf lines that connect the same MW on the ladders on either side of the gel. 3) And where do I put the last Rf line, and how would I edit it to capture the gel distortion? 
Thank you for any help you can provide! I can also upload one of my gel analyzer files that I have been attempting if that would help. 
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Thank you for your response! The manual does not have enough detail for me to understand where exactly the Rf lines should be placed. I am unsure of where to draw them, and their placement changes the length estimate quite a bit (depending on how I draw them). 
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I did several optimizations on my agarose gel electrophoresis for the DNA ladder. The size of my DNA ladder is 100bp-10kb.
1) At first, I used 1.0% agarose gel, I tried to load the sample into the comb with 0.5ul loading dye + 0.5ul DNA ladder. I found that it is quite fine and I still have smiley band.
2)  Second, I  used 1% agarose gel, I loaded the sample into the comb with 0.5ul loading dye + 1.0ul DNA ladder. Although the bands was quite thicker, unfortunately, those smiley bands were still there. But the smiley was getting faded away whenever the ladder went to the smaller size. 
Is that a normal situation of using a big size of DNA ladder? Or Is still there any possible way to troubleshoot?
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Hi and good day everyone.
I am really sorry for a late reply. Thank you very much for getting back to me. I am really appreciated all the answers. I did tried it before with low voltage ~70v for 2.5 hours. I got the band. I did run two gel. In the first gel, the band was nice. But in the second gel, it was not as the bands were slightly wavy. From my observation, I found that the first gel I tried to pour it gently once it was getting warm, The second gel, I pour it into the casting gel even it is still hot. I did read on several troubleshooting on it. One of that is if we pour the gel while it is hot, we might have  a greater chance to have a not well-formed agarose gel. Later I will run the ladder along with my samples. I hope that everything goes well. Thank you very much all of you :D
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I have 600bp long DNA, digested with Restriction enzyme to create 2 fragments one is 500bp long and another is 100bp long I want to see both the bands, so what percentage of  agarose gel should I use?
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Hey  Jain
Look for PDF attached PLZ
Good luck
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I have extracted the bacterial genomic DNA using Roche High PCR Template preparation kit. After elution with elution buffer, I had a white pellet like stuff! I inverted the tubes a couple of times and it just dissolved! The centrifuge balance tube (water) also had this white stuff. I repeated the extraction but this time eluting with water and I had the white stuff both in my centrifuge balance (just water) and eluted DNA. I was wondering if anyone had a similar experience? I don't think it is salt because my balance had just water in it.
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I thought about it, but my colleague told me he dint have any such problem (I used his kit for extraction). I did a phenol:chloroform extraction followed by ethanol precipitation. Still I can see traces of the stuff. I am planning to do a PCR tomorrow. Hopefully, there will be no issues.
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We used a small amount of our digest (5ul out of 100) and ran on the gel to confirm the digests success, when the rest of the digest sample was quantified with nano drop however, no DNA could be measured. Ethanol precipitation didn't work either. Any clues to why this is?
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Also take great care when using nanodrop ( or any spectrophotometer) to ensure that the machine is zeroed correctly. You must use a zero blank that exactly matches your dna solvent .eg if your dna is eluted in tris buffer you cannot use water to blank /zero the nanodrop or vise versa especially with small amounts of dna. I have had negative amounts of dna by wrongly zeroing a noanodrop for pcr product
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Is there other way to measure DNA length rather than PCR - Gel electrophoresis ?
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As your question is not very specific, I agree with all the answers given above. However, I also urge you to look into the link attached that discusses the measurement of DNA length based on weight and concentration of DNA. Hope it gives you more options to work with!
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Hello
I extracted Chlamydomonas genomic DNA. and i checked gel electrophoresis. 
But i can't find genomic DNA in gel. and it remained smear.
i didn't treated RNase.
Can you check my protocol?
1) add 500uL TEN buffer.
2) resuspend, spin 10sec. and aspirate off supernatant.
3) resuspend cells in DW on ice and add 300uL of SDS-EB buffer, voltex.
4) Extract once with 350uL phenol/CIA(1:1) for few min by vortex, separate phases by centrifugation for 5min. transfer aq. to a new tube.
5) Extract once with 300uL CIA(24:1), centrifugation, transfer aq. to a new tube.
6) Add 2 volumes abs.ethanol, incubate on ice for 30min, centrifuge for 10min, wash pellet once with 200uL 70% ethanol.
7) Dry pellet and resuspend in 30uL DW.
what is problem in my experiment??
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Hi Dear Jun-Woo Lee
I think, in your electrophoresis, smear density is very high.  You once repeat the test again with smaller amounts of DNA samples and with high concentrations of agarose gel.
Best, Roshdi
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I attach a photo of my agarose gel.. As you can see the DNA ladder runs like "all toghether" (sorry for bad english). I followed the manufacturer recomendations (1,5% agarose, 90mV, 1 hour)... The ladder goes from 100 to 1000 bp.. what am i doing wrong? 
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I definitely agree with the above comments. To add up to them just make sure that the buffer you use in the gel tank is not very old too. Old buffer can cause poor resolution as well.
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i am running a dna sequencing reaction in a 40 cm long gel but i am  not able to get crisp bands.bands seems diffuse. running condition 1500 V / 1x TBE buffer. oligo length 24 base 5 end FAM albelled .I am attaching a picture below.what would be the possible cause.
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You can follow Prof.Dimitar suggestion but You can perform with FAM labelld oligos also.
Instead of alkalineloading buffer you can try 2X loading buffer(90% formamide, 0.5% EDTA, 0.1% xylene cyanol and 0.1% bromphenol blue)
Before loading the samples pre run for 30 min-60 min.
Deanture the samples before loading usnig 2X loading buffer at 80-90° C for 10 min.
Wash the wells before pre run and loading samples to remove urea.
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I have double digested a vector having size 8 Kb and Insert 100 bp (0.1 Kb). What percent Agarose gel I used use to see both band clearly?
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The problem is not the percent agarose - you can 'see' just about any size band in any gel. The agarose  % is varied to optimize resolution in order to be able to estimate the size. The problem is that your smaller fragment is only 1.25% of the mass of your larger fragment. In order to be able to visualize the smaller band, you will have to load at least a couple of micrograms of digested vector (= 25 ng of smaller fragment). You will see both bands but the larger fragment will be a massive blob. If you really need to verify the sizes of both fragments, you will need to run 2 gels.
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I eluted my amplicon from gel and checked its concentration after elution (column based kit) on 0.8% agarose gel. In the eluted product I found another band above my amplicon, which is very unusual as although it had some primer dimers, but no bands above my PCR product and also, I carefully cut out the band of my desired size only. Strangely, it appeared only after elution. What could it be? Although the band is very faint in the gel, will it be safe to use eluted product for downstream processes?
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 Dear Margison,
1. True, I have used a circular plasmid as my PCR template and have used less than 100 ng of it, which I assume is not an unacceptably large quantity. I really don't know what large quantities most people in this forum use. 
Anyways, I got some help from another forum. People facing similar problem may find it useful.
  • asked a question related to DNA Gel Electrophoresis
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I am trying to amplify a fragment of CMV genome from a dried blood spot (Guthrie card) but all I see in agarose electrophoresis gel are primer dimers, no expected band even in positive control.
It is a nested PCR technique, it has been used hundreds of times in our laboratory but now it seems it is not working anymore. The master mix was already made by a colleague who optimized the tecnique, it worked a couple of times but suddenly no amplification fragment is obtained.
I ordered new Taq polymerase, I made fresh new master mix, I repeated DNA extraction but nothing seems to work.
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