I had done PCR in two batches. I had casted a agarose gel using two combs. I had loaded few samples from the first batch (most of them had been already visualized and had given good amplification) and then ladder and the few samples from second batch in the upper well. The remaining samples of second batch were loaded in lower wells of same gel.
After visualizing the gel under UV no bands were seen in the upper wells except the ladder while good amplification was seen in lower wells.
Why it occurred?
Have you put loading buffer in all of your samples? It's quite strange if you see the bands in the lower wells, and these are exactly the same ones as the ones loaded in the upper wells. I would suggest to repeat again everything, maybe some issue during pipetting...Good luck!
Hard to say without a picture of the gel. Is the issue solved by incubating the gel for a few minutes in running buffer + EtBr?
I had used same load buffer in all my samples and even in the ladder. I even soaked the gel in running buffer + EtBr. But result was the same i.e. NO bands in the upper half.
sir, please specify that have you used same type of primer and same source of DNA in all case, because it may be that one of your primer have not shown any amplification actual and 2nd one is better working possibly....
Usually, such happens when the electrodes are bad. However, since the upper part is not showing any visible bands, it could mean that there is no amplification. This is because the ladder and the lower wells separated very well according to you.
Dear Karen,
I had used run two PCRs in different batches using 20 ng DNA from different patients. ( One with 70 samples and other with 85 samples). Out of first batch 60 were earlier visualized in different gel and they had given good amplification.
In this gel I had loaded remaining 10 samples from the first batch followed by ladder and remaining the samples were from second batch. Gel is not showing any amplification in the upper half except ladder; while band from second batch PCR have given good amplification in lower half.
Dear Anyebe,
The Instrument used for electrophoresis is quite new. ( less than one year). If electrodes were bad I guess there would be no bands in lower wells also.
Suresh,
I think you must upload a picture of the gel for quicker resolution of your problem. People can simply look at the gel and give you their views.
- Badges
- Science method