Hello,
I am new in PFGE and started by checking Lambda ladder electrophoresis accordingly to BIO-Rad recomendations:
-CHEF DR III
-gel in 0.5x TBE,
-recirculated at 14 °C.
-Run time was 22 hours
-Voltage 6 V/cm
-swith time 50 to 90 seconds
-included angle 120°.
-Initial current before placing the gel was 134, at the end of electrophoresis it reached 170
There were 4 lanes with lambda ladder, stained with ethidium bromide. Results attached. Was the run time too long and marker mooved out of the gel?
I work with an older system (CHEF Mapper XA) so I don`t know how much my experience translates to your system.
* Gel size/percentage: check if you have the same gel size as in the recommendations. If you have a small gel maybe the running time is to long for it. If you have a lower percentage gel the DNA can migrate faster.
* When the temperature rises while the run i see similar gels. We have an old machine and the whole system is pretty sensitive so the cooling sometimes stop working. Be aware not to switch on the cooler without the pump running otherwise the tubes can freeze up inside the the cooler, preventing the circulation and so the cooling. Let the system run to precool without the electrophoresis started. I have the feeling (not really checked) that our system doesn't cool if heated over room temperature - so dont let it get warm. The pumping rate at our machine needs to be at 70 for efficient cooling - to fast flow leaves little time for the buffer to cool inside the machine - to low flow leaves the buffer to long in the chamber to heat up.
* Your currency is a bit higher than mine. I also use 0.5xTBE and I have a currency of ~100-120 mA (BUT this could be a machine difference). The currency depends primarily on your buffer-concentration and should stay more or less stable through out the run (a little increase seems to be normal). Make always fresh buffer, use at least milliq water to prepare the buffer and wash out the chamber after each run.
I have some runs which failed. But most of them worked. I never really got to the bottom of the problem. Maybe the machine is just old. But if you work with plugs you most likely will not need the whole plug to load - so store the rest to rerun if needed.
Good luck!
Are you certain that you have intact high MW DNA that you are starting with?
As far as i can tell cooling system works fine and it kept 14°C. Now I will try to first pour the gel and then insert the plugs, maybe this will change something
Hello,
After many attempts I managed to solve some of the problems. Right now, Lambda ladder is quite good (although not fully separated probably due to to short swith time), but the sample bands are smeary, tick and distorted. Current electrophoresis conditions were:
Switch time 6-36s
Voltage 6V
Angle 120
Buffer temperature 14C
Run time 22 h
I have tried adding slimmer slices but it didnt improove anything. I would be realy grateful for any suggestions, because im running out of ideas.
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