Hi Satej,

Some very common mistakes that can cause smearing for the PCR product are listed below:

1. Improperly prepared gel:

If the gel is not poured correctly, it will not polymerize or solidify evenly, thus causing the molecules to smear.

2. Overloading the Wells:

If the wells are filled too much, or if the sample is not properly diluted, the excess sample may smear across the gel. In addition, if the gel is moved after the sample is placed in the well, it can cause the sample to spill out of the well. This can also cause smearing.

3.Improperly Prepared Sample:

When preparing the sample of protein or nucleic acid, it is broken down using an enzyme that cuts the molecule in specific locations (Restriction Digestion/). if this is not done correctly, the enzyme may break down the molecule too much in an uncontrolled way, resulting in smearing. In addition, choosing an incorrect buffer or temperature may also cause the enzyme to function improperly, causing smearing and last but not least

4. Contamination:

If a DNA sample is contaminated with a protein that can also cause smearing.

This is a very common problem for the agarose gel electrophoresis and you can find lots of literature else where.

Good luck!!

Hi, you observed smear during your run or did you have a smear after the run (no clear band)? Cheers, Nadine

Hi Satej,

Some very common mistakes that can cause smearing for the PCR product are listed below:

1. Improperly prepared gel:

If the gel is not poured correctly, it will not polymerize or solidify evenly, thus causing the molecules to smear.

2. Overloading the Wells:

If the wells are filled too much, or if the sample is not properly diluted, the excess sample may smear across the gel. In addition, if the gel is moved after the sample is placed in the well, it can cause the sample to spill out of the well. This can also cause smearing.

3.Improperly Prepared Sample:

When preparing the sample of protein or nucleic acid, it is broken down using an enzyme that cuts the molecule in specific locations (Restriction Digestion/). if this is not done correctly, the enzyme may break down the molecule too much in an uncontrolled way, resulting in smearing. In addition, choosing an incorrect buffer or temperature may also cause the enzyme to function improperly, causing smearing and last but not least

4. Contamination:

If a DNA sample is contaminated with a protein that can also cause smearing.

This is a very common problem for the agarose gel electrophoresis and you can find lots of literature else where.

Good luck!!

@Nadine

Hi, the smear was seen after the run. There were no clear bands just the smear

@ Amritlal Mandal

Hi, Thanks but I dont think one of these could be the reason. The gel was proper, no overloading, it was the PCR product so restriction is rare.

Hi, Amritlal still gave you some reasons. Here are some more:

The DNA was degraded. Avoid nuclease contamination.

Improper electrophoresis conditions were used. Do not allow voltage to exceed ~20 V/cm. Maintain a temperature

@Nadine

Thanks, these all precautions are for avoiding smears in DNA gel but isn't there anything different when we run PCR product

Hi, do you have check the primers befor perfor PCR? and in particular, are you sure about the efficency of your PCR reaction and specificity of your primers?

@ Michele

The primers that i used have worked for other samples

Did you check the quality of your DNA? Which DNA concentration did you use? Are your PCR reagents okay? Did you use filter tips?

@Satej,

I just wanted to summarize the possible reasons that usually affect agarose gel and may cause smear when PCR products are run. It is your responsibility to detect the problems and troubleshoot.

@Nadine,

Thanks for sharing many important information regarding this issue that will definitely help some one to troubleshoot.

One important addendum that I should include:

Primer dimers (PDs) formed during PCR run is a common finding which be visible after gel electrophoresis of the PCR product. PDs in ethidium bromide-stained gels are typically seen as a 30-50 base-pair (bp) band or smear of moderate to high intensity and distinguishable from the band of the target sequence, which is typically longer than 50 bp. One approach to prevent PD formation consists of physical-chemical optimization of the PCR system, i.e., changing the concentrations of primers, magnesium chloride, nucleotides, ionic strength and temperature of the reaction which will possible affect target and PCR reaction in terms of efficiency of amplification.

As a general info, there is an old thread available in RG addressing this issue.

Any one can look at

https://www.researchgate.net/post/PCR_smear

@Nadine, Amritlal

Thanks. I will try as suggested. Will get back if the problem persists.

Hi Satej

Referring to the cause is due to possible nonspecific amplified during the PCR reaction. You could try using a Toychdown to try to increase the specificity of your primers in the reaction.

I would suggest trying optimization of this DNA template. You mentioned that this primer pair worked well with other samples, so the primers should be Ok. Often, the smear of PCR reaction is due to too much template. You can prepare the templates at ten-fold higher and lower than the original concentration. Good luck!

Hi Satej,

Some very good ideas on issues that could cause smearing on an agarose gel have been provided. However, one thing I did not see mentioned was whether you used a ladder or PCR product standard that you know should work. This might help you differentiate whether the smear is caused by the agarose gel, conditions of electrophoresis or because of the products you are loading. If you have a PCR product in stock that you know should have clean bands, this should make a good standard for comparison.

Hi

First of all you need to make your experiment and experimental area contamination free. Check your PCR reagent properly if they are working perfectly then chek your elctrophoresis buffer, power pack and PH of buffer. Some time we take care about the PCR reagents only but we have to be cautious about the power, for example overheating through improper power may also degread your PCR product and create smear. However, when doing PCR optimization of PCR regents is necessary to reduce the smears as well as for better resoulution between the fragements..

Hi,

Use correct gel concentration and EtBr concentration. If possible purify your template DNA once. Good luck.

Even I have the same problem. The primers absolutely work well with other samples but only smear is seen with no bands when i use my samples. what could be the possible troubleshoot I can do.? please suggest me

Hi,  I am also facing the same problem. In my case, I got very distorted  and smeared bands on gene ladder and samples when i prepared 1.5% and 3% of agarose gels. 

Would it be the same reason as everyone mentioned above?

Can anyone  help me with this issue?

Thank you in advance.

Hi,

If the smearing occurs in Ladder as well then there is some problem with your gel. May be the temperature increases during the run.

Hello,

I also have problem with smear in my gel. I have read all the advices above but still dont know what I am doing wrong. In attachment there are my gels, one the good one and another is bad. The conditions were same (different samples).

It is 3,5 % agarose gel stained by Ethidium, 210 V (ELFO for fast running).

Thank you for your time and advices in advance.

Hi,  the very minor problem appearing and difference in between two images is the preparation of agarose. in the 1st image it is good and 2nd is having casting problem where it was not consistently formed. the only indication for the difference is ladder. the wavy bands in ladder. where as in the 1st image they are very straight and clear. need not to bother much.

Well I have problem that I can not be sure, that I read the genotype properly. As you can see the heterozygote samples are not clearly visible and could cause number of mistakes.

Do you have any idea what is wrong with the gel? I mean why the gel is not formed in right way?

And at the 3. picture I don´t know why the 1.,3.5. etc samples are fuzzy. Could it be overloaded?

Check the pH of your TAE or TBE buffer prepare either 1X or 0.5X (use same strength buffer for both gel preparation and electrophoresis)

Is that a 100bp ladder? 

Prepare required percent of gel, in your case 3.5 to 4% for clear results and conclusion. 

If you use a microwave oven for melting the gel, to the weighed agarose powder add approx. 5 to 10% of extra than needed working TBE or TAE before melting. 

Always let the gel be molten thoroughly for several minutes. I melt my 4% gels for about 7 to 10 minutes with few periodic swirls. You must see a clear solution of agarose with out any moving immiscible transparent particles. 

The major problem making the 3% or 4% gels, gel gets overflown. Being very cautious to this and patience helps out.

Even before pouring for casting you gently swirl to make sure that it is clear and uniform and dont bother about bubbles, they will always be on top of gel and never interfere the band separation. 

If you pre stain your gel, need not to wait much, add your EtBr solution into gel and swirl thoroughly and immediately pour into trays. Put your comb then.

Lower your room temperature and give enough time for gel to form nicely and retrieve the comb gently. Immediately place your gel to unit. 

You may give high voltage but see that ur gel is not over heated by that. Use lower room temp. else put some ice pack beneath the boat if possible (sometimes this creates problems too). 

Cheers

Thank you so much for your advices.

I use 50 bp ladder. I will check the buffer pH but my colleagues does not have any problems and they use same equipment and solutions.

I gave a time to the agarose to ,,blow up" and I left it on the mixing desk about 10 min. Then I boiled it in microwave oven for approx. 5 min. And even my colleague checked that the agarose is melted properly and still the picture was not good.

Use buffer kept in fridge for melting the agarose. Add the weighed agarose intto that cold working buffer solution and give it a swirl for few times. Let the agarose particles distribute through out the buffer and then keep for melting and be sure with molten agarose for thorough homogeneity. 

Hello,

my problem with gels disappeared. I am not exactly sure why, because I do everything in the same way I did before. Only change is that I started to mix the agarose during cooking roughly. So maybe I had some not perfectly melted agarose on the edge of the glass and then maybe these rests of agarose damaged the gel.

I just want to tell the others if they have same pictures I did to try just not to be gentle to their gels :)

And I also want one more thank to everybody who tried to help me.

I have a PCR product specific, but in the agarose gel 1% dyed with 2ul (10mg/ml) of ethidium bromide, but gel presented as form dragged in up and in down in the with a band specific of PCR product. What can be?

Hello,

This is my first time ever running gel electrophoresis and i would like to know whats my mistake here? The gel isn't showing at all in the picture. 

Antônio José Rocha, did you solve your problem?

Hello fata ali, 

First, stain the gel properly, mix the molten agarose with stain uniformly. Try a low % gel.

load and run a dna step up ladder or any known good quality, intact dna solution and electrophores for some time and transilluminate . If you could see the intended band, you did it great.

Good luck.

Thanks a lot, this quite a problem that currently disturb my work.

I agree  with Amritlal Mandal 

this is some common mistakes that can cause smearing for the PCR product 

1. Improperly prepared gel:

If the gel is not poured correctly, it will not polymerize or solidify evenly, thus causing the molecules to smear.

2. Overloading the Wells:

If the wells are filled too much, or if the sample is not properly diluted, the excess sample may smear across the gel. In addition, if the gel is moved after the sample is placed in the well, it can cause the sample to spill out of the well. This can also cause smearing.

3.Improperly Prepared Sample:

When preparing the sample of protein or nucleic acid, it is broken down using an enzyme that cuts the molecule in specific locations (Restriction Digestion/). if this is not done correctly, the enzyme may break down the molecule too much in an uncontrolled way, resulting in smearing. In addition, choosing an incorrect buffer or temperature may also cause the enzyme to function improperly, causing smearing and last but not least

4. Contamination:

If a DNA sample is contaminated with a protein that can also cause smearing.

This is a very common problem for the agarose gel electrophoresis and you can find lots of literature else where.

Good luck!!

Waleed

I think PCR has not worked, bands visible are probably primer dimers. If the ladder is not resolved properly then there is problem with the gel as well.

Hi,

There are many informative answers.

I hope you PCR product issue is solved.

I would like to add that there might be contamination in the 1XTBE buffer that you are using, use freshly prepared buffer, dont heat the agarose too much and also cleas all the glass wear before preparing agarose.

Thank you.

Hello everyone,

I also got the smear band in samples, not in DNA ladder on gel when I extracted Enterococcus isolates. I think I have problems with DNA extraction especially concentration of enzymes that I used. I used 10mg/ml lysozyme (final concentration - 2.5mg/ml), 10mg/ml Proteinase K (fincal concentration - 366 ug/ml), 20% SDS (final concentration - 0.9%) in 10mM Tris HCl,25mM EDTA TE buffer and incubate 55'C for 30 minutes. After that, I extract DNA with phenol chloroform method.

Could you check my concentration of enzymes are suitable for Enterococcus? Please could you share your experience of genomic DNA extraction from Enterococcus bacterial culture?

Thanks.

Hi All,

Maybe you need to check

1. DNA quality

2. Concentration of MgCl2 and DNA template

All the best.

Hello Dr. Satej Bhushan

you can read this

1- PCR Troubleshooting

http://www.aun.edu.eg/molecular_biology/pcr/L_schow_PCR_2007_2_black_white[1].pdf

2- Why do I get smeared PCR products?

https://www.qiagen.com/kr/resources/faq?id=4eb03cc8-4623-4e9e-96b2-6a4c17c03c58&lang=en

It is obviosuly target gene and primer specific but