Hi Alexandra

'A picture is worth thousand words'

It was nice to see the photograph...than actually visualizing the problem you are facing....First of all there are no bands on the lower side of the gel....only smear is visible as pointed by researchers above....I have few suggestions

1) First of all your DNA looks fine for most of the routine applications.....there is some amount of smear...but it should not be much of a problem

please note that the smear is visible mostly in the lanes where 'loaded sample' is in large amout......low quantity bands looks 'clean''....But I am sures if you load more amount of DNA.........in low loaded sample...smear will be visible even in those

2) some amount of smear will be always there with routine methods of DNA isolation....if you really need high quality DNA you will have to use some special protocols

3) If you still want to reduce the smear in your protocol.....take care of few things

a) Use liquid Nitrogen during extraction if possible.....as at low temp...DNAses will be inactive

b) Try to use denaturing agents such as urea (SDS or some other equivalent) to denature the proteins/enzymes during extraction

c) Avoid harsh steps that can cause shearing....some are suggested above...like use of wide-mouth tips, invert the tubes for mixing rather than vortexing etc.

d) use RNAse for degrading the RNA as this can also be seen a s smear..

e) Last but not the least....'do your extractions quickly but gently'....

This actually seems to be a smear. During DNA extraction, degradation of DNA may occur and this gives such smear. Your other DNA bands are good, which means your reagents are not a problem. The only problem seems to be 'handling error'. Try to extract DNA once more of smaples that gave smear.

make sure your tubes tips and other instruments are DNAse free and you will get an excellent results.

Genomic DNA being physically fragile shears due to pipetting or vortexing. DNA molecules> 150 kb are prone to breakage by forces generated during isolation.

Solution- Cut the base of the tips and use them. Do NOT vortex.

Another possible source of error could be DNase contamination in tips or tubes.

Solution- Sterilize them properly before use.

Thank You for correcting me Nancy.

Its cutting the tip of the tips that I intended to say.

:)

Such band appears bcoz of Dnase activity on DNA..

All the comments are appreciated as I read them all and reformed myself too. Thanks to All

if Impurities like RNA, Protein, and DNA shearing , even high Voltage can cause such smear.

Hi Alexandra

'A picture is worth thousand words'

It was nice to see the photograph...than actually visualizing the problem you are facing....First of all there are no bands on the lower side of the gel....only smear is visible as pointed by researchers above....I have few suggestions

1) First of all your DNA looks fine for most of the routine applications.....there is some amount of smear...but it should not be much of a problem

please note that the smear is visible mostly in the lanes where 'loaded sample' is in large amout......low quantity bands looks 'clean''....But I am sures if you load more amount of DNA.........in low loaded sample...smear will be visible even in those

2) some amount of smear will be always there with routine methods of DNA isolation....if you really need high quality DNA you will have to use some special protocols

3) If you still want to reduce the smear in your protocol.....take care of few things

a) Use liquid Nitrogen during extraction if possible.....as at low temp...DNAses will be inactive

b) Try to use denaturing agents such as urea (SDS or some other equivalent) to denature the proteins/enzymes during extraction

c) Avoid harsh steps that can cause shearing....some are suggested above...like use of wide-mouth tips, invert the tubes for mixing rather than vortexing etc.

d) use RNAse for degrading the RNA as this can also be seen a s smear..

e) Last but not the least....'do your extractions quickly but gently'....

Your samples DNA is sheared and that's why you got smear of DNA .check your extraction step.

It looks like you have samples from various species of fish. If the samples were not fresh, its also possible that DNA degradation may have occurred in the sample itself and was present before the extraction procedure began. Were the samples all from the same tissue? Some tissues may have more nucleases than others or this may vary between species. Also the size of the tissue sample might make a difference. If you add too much tissue to the extraction, then you may get more DNA, but nucleases will not be so rapidly inactivated.

The band worrying you is close to the bromophenol blue dye front, I suppose. This could be RNA or some SDS-nucleotides-coplex. Treat with RNase and see whether it disappears.

Always run a ladder. Anyhow, that looks like classic DNA degradation. Maybe you have some DNAse contamination somewhere in one of your buffers or mishandled somehow. If you look closely there is smearing in every lane so run a ladder and if that has smears then it might be something in the agarose or something in the preparation of the gel. Always run standards.

Hello,

I've been there and in my case, the DNA was degraded. The best solution was to extract again.

good luck

Hi Alexandra,

Which extraction method do you use? I believe that your samples are contaminated with proteins, because you have precipitated in the wells.

A simple protein extraction with phenol/ chloroform and precipitation with 2-propanol may resolve your problem.

Good luck

Hi Leonel,

please explaine me how you can see the proteins precipitated in the wells. What kind of gel staining must be used to see precipitated proteins, or why, how they can be seen. I have never heard or read of it before, but could be a very useful information when DNA extraction is not too succesful. Could it be the DNA and protein complex which is precipitated and stays in the wells?

Thank you in advance.

The precipitated materials do not move much from wells. The precipitation might interfere as some DNA will be bound to it resulting in low yeild. Use of proteases from early stages might help to some extent, particularly proteinase K.

The marked lanes contain DNA that has been sheared. The main identification of sheared DNA in an agarose gel is a long smear towards the end of the gel starting from the lower end of the DNA band. My suggestions for obtaining good quality intact DNA-

a) use fresh samples that have not been stored for long under refrigeration.

b) avoid steps like vortexing and frequent pipetting that could shear the DNA.

good luck!!

Did you mean bands or smear? Because i also faced the same problems but it is because of my extraction method where i used high concentration of salt in my extraction........what kind of method that you used?

Do you have a good quality of extrated DNA? I have understood that when this happens could be a problem of DNA degradation, so tha is why you see bands of lower molecular weigh! How do you treat your DNA before the gel preparation?

You can have problems with purification procedure too!

Be careful about mistaking "Freddy the Ghost Band" for an actual cloneable fragment. See http://ac.els-cdn.com/S0968000496300418/1-s2.0-S0968000496300418-main.pdf?_tid=7a2369520f7e4569d971112131bfadd2&acdnat=1339713229_7c6d85218c71ed8f3e6b5618f49584bc.

Hi Alexandra (and others),

This is long ago now, so I guess you have sorted it out. Looking at the replies above, though, I would stress that IF you have extracted all samples that are run on the depicted gel together, then there should be no problem with you extraction methods.

The smear you see, just as explained by several others, represent degraded DNA, i.e. shorter fragments. I would argue that this degradation likely has happened in the non-extracted sample, due to length of storage and storage conditions (or conditions of the individual which was first sampled). Therefore, there is not that much to do about it.

For regular PCR's this shouldn't be a huge problem as long as there is also some high molecular-weight DNA (or really as long as there are fragments that are longer than that which you wish to amplify; try, or use a ladder on the gel with gDNA). For some other techniques, which depend on high quality non-degraded DNA, you may have a problem.

Cheers,

Martin

I am also getting trouble while loading DNA, I got clear band near the well but degredaded types band were obtained appeared again and smearing was seen, that happened in all loaded samples, could you please suggest be the proper answer??

I used CTAB extraction protocol 

My gel colour appear pink instead of white in agarose gel. I am using 7 ul ethidiumbromide in 2 percent gel.can any one can help me?

The pinkish color is because you are using quite a lot of ethidiumbromide for 2 percent gel. I would try just 2 ul of ethidiumbromide. Though using extra ethidiumbromide doesn't necessarily make any problem.

Thanks Mabi Nobari for giving me such good suggestion .