I run the Gell for DNA of some of samples. However some DNA bands were thinner some were more thicker in size and some of them were like smear from top to bottom. What could be the reason?
Different band intensity corresponds to the concentration. Smear indicates DNA degradation probably due to nuclease contamination in the sample.
Dear Aqleem,
differences in band intensity might have been caused by differences in the use of alcohol [like isopropanol if you are using a kit] .
Regarding the smear DNA you see in the gel image, there are many factors that can influence such outcome, which could be:
1- High voltage use and low Agarose concentration.
2- Using expired or poorly mixed phenol.
3- Extensive use of absolute Ethanol [less likely].
To solve this issue consider changing voltage first and see what happens.
Regards,
It is hard to say without a picture of the gel but smeared genomic dna is not a problem.It is quite normal for shaking to break up long strands of dna as well as some dnase activity. PCR will be unaffected by randomly broken dna so long as the pcr size is smaller than most of the degraded dna. Different amounts of dna can often be caused by incomplete precipitation or incomplete breaking of the cells producing the dna sample. For very small amounts of dna more can be precipitated by adding a co precipitant like glycogen o bring down more dna
Dear Friend,
It would have been better if you would have uploaded the gel picture. But still then know that smears in the DNA samples show that the DNA has been sheared. To avoid shearing of the DNA avoid vigorous thawing after removing the samples from -200 C. Do not vortex for thawing. Let the vials thaw by defrosting at the room temperature without shaking. Of course you can invert them by holding in hand gently. Note that while DNA isolation there is only one step that needs vigorous vortexing i.e. the first step (after adding CTAB buffer to the crushed sample and keeping in water bath at 65 0C ). Other subsequent mixing steps has to be done very gently to avoid shearing. And secondly the thickness and intensity of the band is based on the concentration of the DNA sample loaded into the wells.
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