Dear Navneeti

Wavy DNA bands on an agarose gel can be caused by:

Gel incompletely immersed in electrophoresis buffer: Electrophoresis buffer should completely cover the entire gel during sample loading and run.

Low sample volume: The sample or the ladder volume should be large enough to fill 1/3 of the total capacity of the well. Large wells should not be used with small sample volumes. If needed the sample volume can be adjusted with 1X loading dye.

Improper electrophoresis conditions: Do not use an excessively high voltage for electrophoresis. Run the gels at 5-8 V/cm. To minimize band curving, use a lower voltage for several minutes at the beginning of electrophoresis. To calculate the optimal electrophoresis conditions (voltage) and to use the recommended V/cm value (which is in many cases 5-8 V/cm, depending on the ladder) one has to: – measure the distance between electrodes (cathode and anode) – X, cm. – and multiply the X value by the recommended voltage (Y, V/cm) – the result (X x Y) is the recommended voltage to be applied.

Bubbles or physical particles in the gel wells or in the gel: Use pure water, clean flasks and clean equipment for preparation of gels. Pour the gel slowly avoiding formation of bubbles. Bubbles can be removed with a pipette tip.

Dried agarose on the comb teeth:  Check the comb teeth for residual dried agarose prior to casting the gel and clean if necessary.

https://www.qiagen.com/ca/resources/faq?id=dfb243d4-eb9a-4e60-bad7-37fe2c7d371d&lang=en

Are you running in TAE or TBE? What is the size of your PCR product of interest? Which size of gel, and running at which voltage? All of these different conditions can lead to differences in the way the gel runs. For a 1.2% gel, you should be visualizing DNA between 400bp and 7kb. If using a mini gel size, try running at 90V and see if that makes a difference (time will have to depend on expected size of the PCR product and the desired separation). In the end, an agarose gel doesn't typically have to look pretty, it is typically just used to see if your desired DNA is present (especially after PCR), so I wouldn't worry too much about a small wave in the DNA band.

Dear Navneeti

Wavy DNA bands on an agarose gel can be caused by:

Gel incompletely immersed in electrophoresis buffer: Electrophoresis buffer should completely cover the entire gel during sample loading and run.

Low sample volume: The sample or the ladder volume should be large enough to fill 1/3 of the total capacity of the well. Large wells should not be used with small sample volumes. If needed the sample volume can be adjusted with 1X loading dye.

Improper electrophoresis conditions: Do not use an excessively high voltage for electrophoresis. Run the gels at 5-8 V/cm. To minimize band curving, use a lower voltage for several minutes at the beginning of electrophoresis. To calculate the optimal electrophoresis conditions (voltage) and to use the recommended V/cm value (which is in many cases 5-8 V/cm, depending on the ladder) one has to: – measure the distance between electrodes (cathode and anode) – X, cm. – and multiply the X value by the recommended voltage (Y, V/cm) – the result (X x Y) is the recommended voltage to be applied.

Bubbles or physical particles in the gel wells or in the gel: Use pure water, clean flasks and clean equipment for preparation of gels. Pour the gel slowly avoiding formation of bubbles. Bubbles can be removed with a pipette tip.

Dried agarose on the comb teeth:  Check the comb teeth for residual dried agarose prior to casting the gel and clean if necessary.

https://www.qiagen.com/ca/resources/faq?id=dfb243d4-eb9a-4e60-bad7-37fe2c7d371d&lang=en

maybe you can regulate lower voltage or change new TAE or TBE solution

Hi,

Dried agarose on the comb teeth is a frequent cause of this problem. Prior to casting your gel, check the comb teeth for residual dried agarose. If not removed, this will attach to the newly cast agarose and fracture the well upon comb removal. This is usually not observable until the gel is on the transilluminator. Additionally, care must be taken during comb removal, particularly with low melting temperature agaroses. Well integrity may be maintained in these agaroses by pre-chilling the gel to 4°C for 30 minutes and/or by flooding the gel with cold buffer prior to removing the comb.

Hi

Thanks to everyone. I am working on all the suggestions.

I am using TBE.

Can anybody tell me the concentration and ammount of Etbr. Can it also be a reason?

Hi

The gel could be stained in a 0.5 µg/ml Etbr solution for 30 min.

Thank you Seied Mehdi Miri .

I am using TBE Christopher Kesthely and all the conditions are same as you mentioned here. I have calculated the voltage by the distance of the electrode but still getting wavy bands.

Hi,

Here I am attaching the photo of the Gel. Please Have a look and give suggestions.

Thank you.

Based on your photos, the upper band looks fine and quite normal. The smaller fragment is a little strange. Make sure that you completely melt all of the agarose in your solution before poring the gel (it will all be boiling), then let it cool for a bit before pouring your gel. As previously mentioned, use a well appropriate to the amout of DNA being loaded (if a small volume, use a small well size). Otherwise, it may be worth it to run a PCR purification before running on the gel as this should help remove some of the background signal that you have in your samples, and may also help clear up the band a little bit.

Misshapen bands (wavy, broken in middle, dumbbell shaped)

I have copied the following for you:

Examples of misshapen bands (wavy, broken, or dumbbell shaped)

When the gel is run fast, the DNA moves more at the sides of the band than in the middle, giving it a shape similar to a dumbbell. This probably is due to pushing the DNA through the gel faster than it can migrate through the gel in a uniform manner, given the drag on its migration by the gel matrix. This effect is greater with loading of larger amounts of DNA, larger sized DNA fragments (which contain more DNA than smaller sized fragments cut from the same plasmid), faster runs, and narrower lanes. As more DNA migrates at the sides of the band, there is more tailing or streaking of the DNA backwards (upwards) towards the wells from the sides of the bands. In severe cases, very little DNA remains in the center of the band giving the band an appearance like a "W" or even appearing to have been split in the middle into two half-width bands migrating in the gel at the same rate. However, the band thickness (viewed from above) usually is not much greater for W-shaped or dumbbell-shaped bands than for rectangular-shaped bands. Though this is a common problem for gels inthis lab, it is not a major problem for data analysis or interpretation. However, when this occurs, the migration distance of such bands should always be measured using the bottom-most point of the band (the part of the band that migrates the fastest). If the band is extremely elongated when viewed from above and is in the shape of a W or is extremely wavy, see the next paragraph about severe tailing and very poor separation.

Thanks to all.By your suggestions soon i will upload a fine picture of gel.

The gel probably has a problem. Also reduce the degree of electrophoresis slightly

For DNA you should use TAE not TBE. Glycerol (which is present in DNA dye) causes "streaming" of the sample DNA to yield U-shaped bands and can react with boric acid ion of TBE to disturb band resolution.

You can the see this paper:"History and Principles of conductive media for standard DNA electrophoresis" by Jonathan R.Brody and Scott E.Kern.

Wavy DNA bands on an agarose gel can be caused by dried agarose on the comb teeth.  Check the comb teeth for residual dried agarose prior to casting the gel and clean if nessary