Hi !
I'm comparing the performance of two different thermocyclers. One of those is a commercial thermal cycler and the other is a prototype. For this test I prepared a 60 ul PCR mix and then separated it into two 30 ul tubes. A 30 ul layer of mineral oil was applied to both tubes at the end of the preparation. As soon as the PCR was finished in both thermocyclers, 10ul of each sample was mixed with loading buffer to put them in the electrophoresis gel (The PCR sample of the commercial thermal cycler is the one on the left, the PCR sample of the prototype is the one on the right The other wells are empty). But at the time of putting the gel under UV light these were the results (see attached images).
I have a couple of questions. Was there any amplification in the prototype thermocycler? Is there any factor that would cause the DNA sample to migrate in the opposite direction?
if your dna is running in the wrong direction then you probably have the black and red electrode leads connected the wrong way round but in the picture you show it is running correctly so the electrophoresis is fine but there is no sign of amplification on the right of the gel
Hi there,
You should always run a DNA ladder in parallel with your samples. Phosphonucleotides are always negatively charged at working pH so there is no way to make them move toward the cathode. Your amplification has failed for the second lane.
I agree with Paul. Your sample didn't "run backwards". The migrated band in the first well and the faint marks from your loading dye prove that your gel was run correctly. You didn't get any amplified product in the second well. The light colored spot is probably just a blob of not-quite melted agarose or a reflection from the light source. Double-check that you entered the program correctly into the thermocycler.
It is possible that as oil sticks to plastic tips then the volume of oil in the 2 tubes was different and if the total volume in the tube that did not work is higher than the other sample then temperature changes will be slower and the annealing and denaturation temperatures may not have been reached so the pcr did not work. Many old pcr machines were deigned for specific total volumes and did not work well due to temperature overshoots and undershoots when the volume was much different from the designed volume
Paul and Katie were right...
Also, there is no chance that you might plug-in the red and the black wires in the wrong way, because you already have one band migrated in the right direction. You may increase the pcr reaction volume, hopefully it will work.
Regards.
Thank you all, I will make some changes to the machine. I was a little confused by the light colored spot on the second well.
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