Wednesday 23rd April 2014
This week began on Wednesday, as I didn’t get back from Oxford until Monday afternoon and I needed some time to sort myself out before getting back to lab work. I decided to spend the rest of the week continuing my work on PFGE and, seeing as the QIAcube had been fixed while I was away, conducting plasmid extraction.
I started the day by preparing plugs for PFGE by restriction enzyme digest. The plugs were incubated in 0.1 x wash buffer for 1 hour. This was then removed and replaced with 1 x NotI buffer for another hour with gentle agitation. This was again removed and replaced with 1 x NotI buffer and NotI restriction enzyme, incubating overnight at 37ºC.
Thursday 24th April 2014
Today began with collecting the bacterial plugs from incubation, removing the buffer and enzyme then incubating the plugs in 1 x wash buffer for an hour with agitation. This was then removed and replaced with 0.5 x TAE buffer. While they were incubating in TAE I prepared and poured a 1% agarose gel.
This gel was then loaded with the plugs and an appropriate PFGE ladder before running overnight using the following settings:
- 20 hours
- 1% agarose gel
- 0.5x TAE
- 40-100 switch intervals per second
- 6 V/cm
- 30 minute stain, 10 minute destain
Based on the last gel I ran before Easter, I decided to reduce the size of the plugs used in order to try and obtain clearer bands. I would also like to use another ladder alongside my CHEF DNA size marker in order to measure the bands present below the 225Kb band on my current ladder. I decided on a CHEF DNA Size Standard Lambda Ladder, which demonstrates bands from 0.05Mb – 1Mb.
I spent the rest of the day trial running the recently fixed QIAcube to ensure that the new O ring was holding the pipette tips on the robotic arm correctly. I then removed my previously centrifuged and frozen Lincoln collection Escherichia coli cell pellets from the freezer to allow them to defrost. While waiting I prepared the centrifuge rotary adapters and re filled the QIAcube pipette tips and reagents.
Each run of 12 samples took around 50 minutes, and I managed to get samples 1-50 completed with no problems. I decided to finish off the rest of the Lincoln Collection Escherichia coli isolates tomorrow.
Friday 25th April 2014
I started today by finishing off QIAcube plasmid extractions for samples 51-.140. Once completed, the extracted DNA for all samples were stored in the fridge until next week when these samples can be run on gel electrophoresis in order to visulise if plasmids are present.
Once my PFGE gel had finished I stained it for 30 minutes, destaining for 10 minutes before viewing under UV. The resulting gel was as good as my last, with slight improvements in regards to the darker banding areas near the 225Kb band. I feel as though reducing the size of the plug used did help a little to clear up the darker regions, however not completely.
Hopefully my new CHEF DNA Size Standard Lambda Ladder will have arrived and I can use it to begin approximating the smaller bands visible. I would also like to make another batch of bacterial plugs, reducing the amount of DNA present so that the darker regions visible demonstrate clearer bands.
Now that all plasmid DNA has been extracted from the frozen Lincoln collection Escherichia coli cell pellets I need to run these through gel electrophoresis in order to visulise if plasmids are present. Once samples with plasmids have been identified then I can get started on PCR! I also hope I will be able to collect water samples from Bradford in the coming weeks and begin a comparable Bradford collection of Escherichia coli isolates.
A Year in the Lab…