i completed a gel electrophoresis and in the first well i have the expected outcome for tRNA,DNA and rRNA but 3 wells over there appears to be nothing. could contamination cause this or what other possibilities
Dear Sam Boca,
Yes, contamination can cause this. As you will see the RNA only if it has amplified successfully, it will not appear in gel electrophoresis if there any anomalies during extraction and/or PCR amplification (such as contamination during extraction, use of improper primers, following inappropriate PCR conditions, etc.). Further, it might also result from the preparation of the gel. For example, we usually use 1.2% to 1.5% agarose, if it fluctuates, it might cause an unexpected result as you. I hope this answer will help you. I wish you success in your experiment.
it would be helpful if you can upload an image of the gel. Maybe the dye you use for staining already moved to far out of your gel so you dont see the upper lanes anymore?
Dear Sam,
In addition to earlier mentioned reasons, possible reason of not seeing any bands-
1. Poor source for extraction
2. Rough pipetting may cause shearing
3. Gel overrun
Do you measure concentration of RNA ? It 's not vialiby for overrum electrphoresis.
Hi, perhaps it's worth double checking the markers/probing of the RNA. As the labeling process may be the fault. Not showing the RNA during the X-ray visualization stage. Or some adverse reaction may have taken place between the RNA and the labeling of compounds, leading to no visualization results.
Shahid Khalid
Wow.... s much of technical stuff...
- Markers/probes, X-rays, adverse reaction, labeling, compounds....
I wish you could provide a more descriptive and detailed answer, so that it would become some fun here on RG.
Given that the first well in your gel gave the desired results you can rule out:
gel overrun
forgetting the EtBr
expired kit reagents (assuming all samples made at the same time with the same reagents)
My guess is that you have a really low yield of RNA from your samples. Have you measured the concentration?
this is because of contamination or because of low conc. of loading dye
Hamzah H.Kzar
Can you explain how and what contamination prevent bands to appear on gel?
And how low concentration of loading would have any effect on the bands?
Abhijeet Singh you can use google, some reading and youtube for these answers. It's easy enough :)
Shahid
Shahid Khalid
I guess you took my post on your answer too seriously. Previous comment basically was saying that you are not correct with whatever explanation you provided to the question. Additionally, if you understand why ask explanation to Hamzah H.Kzar 's answer, you would not have suggested to check/search on google.
Hi Sam Boca,
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