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Hey Everyone! This is my first post on here, but I will be posting once a week on a Friday to give a summary of each exciting week in the lab.
Amy and I are working on the molecular characterisation of clinical Propionibacterium acnes strains by pulsed-field gel electrophoresis (PFGE) along side Mike. This week we have been concentrating on perfecting the method and settings that will give us the best DNA separation.
Monday we started off by making our TAE buffer stock and EET (used in making of the plugs we will be using for the electrophoresis) and re-familiarising ourselves with the never ending units and measures that are involved in science. I must say that my Chemistry A-Level came in useful for once (never thought I would utter those words). After our buffer was autoclaved and ready to be used we ran our first gel (exciting stuff!). We ran 9 lanes: 2 Lambda Hind III, pure B1 DNA, pure PUC BAM H1, pure PUC ECO R1, pure PBC SK+ ECO R1, pure PBC SK+ BAM H1, raw PUC and raw PBC,at 110V for an hour. We then stained the gel with Ethidium Bromide and observed it under UV light. The runs looked slightly messy and the ends of the bands were slightly raised on the sides, we suspect this was due to a temperature increase when running the electrophoresis.
Tuesday we ran our first PFGE with lambda DNA and BioRad and NEB plugs of the same strain of yeast. The cooling unit was not working so we were running the gel at 26 degrees celsius and over. we concluded that when we run it again we should chill the buffer in the fridge beforehand in order to start at the lowest possible temperature, if that also did not work we would have run the whole thing out a fridge. We set it up to run for 3 hours at 1 second switch intervals and observed the results the next day.
Wednesday we observed the gel we had run on tuesday and concluded that the switch interval was too quick as we saw smears instead of clear bands of separation, so we changed the switch time to 45 seconds keeping everything else the same. We ran the gel for 4 hours and observed it in the afternoon. We saw some separation from the yeasts however we didn’t run it long enough to show further separation of bands further down the gel. Also some of the DNA had not left the wells and therefore had not entered the gel. We made a plan to repeat the gel in exactly the same manor on Thursday, however we would run it for longer and seal in the plugs with agarose gel. We also decided to run the PFGE at 15 second switch intervals for 3 hours to make sure the DNA enters the gel and then run it for a further 21 hours at 45 second switch intervals.
Thursday we came in and made our buffer and left it to chill. Then we made the agarose gel, which took 3 attempts as our morning brains took over and we kept making silly mistakes. I forgot to put the plate in the first time, then we didn’t tighten it well enough so the agarose was going everywhere! We then snapped out of it by half 10 and managed to set an agarose gel correctly. We then proceeded to put the plugs into the wells and sealed them with more agarose. We then ran our PFGE for 3 hours before reprogramming it and leaving it for 21 hours.
Friday we came in and filmed our Vlog which was rather exciting! We have a very promising gag reel so far. We then stained and took a picture of our gel ready for it to be analysed with mike on monday!
All in all it was a good week in the lab. Looking forward to next week! I will soon work out how to upload photos to these blogs so I can put up some pictures of our beautiful gels.
See you guys next week!