DNA Gel Electrophoresis - Science method

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?

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.

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.

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

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

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?

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！

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

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!

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!

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.

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

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? 

Currently, i am thinking about GelAnalyzer software. Please help me with the analysis and band scoring. I have used 100 bp ladder.

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

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!

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

Whether I should use Scion image or Imaage J software for DNA bands analysis?

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?

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!

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!

Can you use e-gel ladders on normal 2% agarose gels?

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

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!

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?

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.

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.

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

I would like to draw a DNA structure (both single strand and double strand).

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!

Need your suggestions...

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?

i had restriction digestion result and i would present it this week is there and advises academically

cheers

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?

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.

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? 

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.

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

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!

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

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!!

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.

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

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...

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.

I have also run the internal control gene. How to normalise, plot graph and in which units to express the data.

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.

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.

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.

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.

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!

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

Do you guys know a cheaper alternative to sybersafe for running a gel?

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

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

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

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

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. 

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?

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?

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.

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?

Is there other way to measure DNA length rather than PCR - Gel electrophoresis ?

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??

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? 

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.

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?

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?

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.