Evening Everyone,
As soon as Me and Jo got into the lab today, we made up 3L of TAE buffer, and left it to chill until we needed it. We then turned off the PFGE, as it had been running overnight, removed the gel and placed it into Ethidium Bromide for 30 minutes to stain. While it was staining we emptied the PFGE of buffer. We then analysed the gel with Mike under UV.
The separations appeared blurry and unclear, Mike told us that this was most likely due to contamination with nucleases, and too low field switching (the time it takes for the electric fields to switch from 1 to 2). We therefore decided to increase our field swtiching time from 1 second to 45 seconds (using ‘Pulsed Field Gel Electrophoresis – A practical guide’ Birren, B. and Lai, E). We prepared another gel, with 10 lanes, containing 3 repeats of BioRad yeast, 3 repeats of NEB yeast, 3 repeats of Lambda DNA and 1 loading buffer as a reference. (Produced in the same way as yesterday). Me and Jo got the opportunity to cut the yeast plugs ourselves today, which was really good! We then started the PFGE at 2.0 V/cm for 4 hours with our new field switching time of 45 seconds. We waited for half an hour, then we turned on the pump, this time was to prevent our liquid Lambda from being washed away.
After the 4 hours, we removed the gel, stained it, drained the buffer and viewed it under UV. This time we saw very dense DNA still within the wells, but fairly good band separation close to the well. Mike suggested that we should seal our yeast plugs with agarose to ensure that the DNA left the wells and began separating. He also suggested that we change our program profile, therefore having an intitial stage for 3 hours at 2.0 V/cm with a field swtiching time of 15 seconds, in order to give the DNA time to leave the wells properly before switching to 45 seconds field switching for the reminder of 24 hours; 21 hours. We then also decided to have a final stage for an hour at 0.6 V/cm, and 45 seconds field switching, until we could get back in Friday morning to check the gel, remove, stain and view it.
Ready to get started tomorrow morning, with our new changes! 🙂