Hey All! 🙂
Didn’t do very much today, as we are trying a 24 hour run with our new modifications we decided on yesterday. Jo and I came in at 9am, and made up 3L of TAE buffer, and left it to chill while we prepared our agarose gel. After we poured it and left it to set (which took a few goes today, as the gel box wasn’t tightened enough and gel kept escaping!) we prepared our samples. We used 2 repeats of BioRad yeast, 2 repeats of NEB yeast and loading buffer as a reference. For this run we also decided to seal the sample plugs within the wells with agarose, in order to promote the movement of the DNA from the wells and into the gel. We used a pipette tip cut with a knife, to give a larger, slanted tip, from which we pipetted agarose into the wells. This worked very well, and we managed to fill the wells easily. We then placed the gel into the PFGE, added buffer and our loading buffer. We turned on the pump to expel any bubbles trapped within the pump/tubes, and then ran the PFGE for 3 hours at 2.0 V/cm with a field switching time of 15 seconds. We decided to do this initial stage to give the DNA time to start moving into the gel, and hopefully allow clearer band separations.
At 2pm we returned to the lab to reprogramm the PFGE to run for 21 hours at 2.0 V/cm with a field switching time of 45 seconds, followed by 2 hours at 0.6 V/cm with a field switching time of 45 seconds. This run should finish at 11am tomorrow morning, therefore the second programm was put in incase we were not quite ready to remove the gel.
Tomorrow morning we will hopefully get to see how our modifications have improved our separations, and if not discuss further modifications for next week. We will also be filming our first video blog!
See you all tomorrow! 🙂