I used 1.5% agarose gel & 1.2% as well with TBE buffer to check DNA fragmentaion in SiHa cells (cervical cancer) after treatment with Cisplatin but I am not getting fragmented bands. I got nuclear fragmentation by Dapi staining with the same treatment.
I have attached the gel image.
Can anyone tell me why I am not getting DNA fragmentation by gel electrophoresis?
What size is the DNA fragmentation you want to detect in agarose gel?
I am not sure but I am assuming shortest fragment can be 100 to 200bp. According to published literature Cisplatin causes apoptosis. I analysed apoptosis by Annexin V and PI staining but as a confirmatory test I want to do DNA fragmentation by gel electrophoresis.
Perhaps you should provide more details about your experiment and information about the gel. Clearly specify what each lane label represents rather than others to guess.
I am guessing that you are expecting to see a decrease in the large DNA band, which will be obvious if you have more than 20% DNA fragmented to smaller size.
As for being able to see fragmented DNA itself, that will be possible only if your experiment causes specific size DNA fragments, as one generates by using restriction enzymes. Otherwise, if random size DNA is resulted by your procedure, you can only see smearing, which will become obvious when a larger portion of the DNA is fragmented. This does seem apparent in some lanes.
Since the size of DNA fragments you wish to detect are small, you can increase the concentration of your agarose gel up to 2%. However, there are other alternative solutions such as using polyacrylamide gel.
As Mahdi said, increase the percentage of the gel. During apoptosis, as a result of endonuclease activity, you will be getting fragmented DNA around 180 bp. Use 2 or 3% percentage gel to get clear visualization of bands
- Badges
- Science topic
- Similar topics
- Biological Science
- Bioscience
- Biotechnology