Tag: TGYE agar

Afternoon Everyone Started off today by viewing our…

Amy Thompson / / Uncategorized

Afternoon Everyone!

Started off today by viewing our latest gel with Mike. Unfortunately our samples ran completely off the gel, and therefore we only saw the areas where the samples had passed. We also saw that the wells were very dark, due to too much DNA still being present within them. We also used the wrong ladder for Propionibacterium acnes. Mike therefore decided that we should run our next gel for 24 hours, at 4V/cm with a 30 second switch interval time, based on our two best previous gels (PFGE 5 and 6). We also decided to cut our plugs into different sizes in order to see how the size of the plug would effect the gel. We cut the plugs into two small pieces, one medium piece and one large piece. We still used samples of both plugs treated with restriction enzyme and without. We also used a reference ladder of NEB yeast.

We then helped Mike to re culture the Propionibacterium acnes by making up TGYE agar, autoclaving it and pouring 15 plates 20ml each. Which i have seen done before, but never done myself, so it was good to have the opportunity to practise! Managed to pour them all pretty well 🙂

We came in at 5pm, to cut off the first well of the Propionibacterium acnes sample treated with restriction enzyme, to see it’s progress. We removed it, stained it and viewed it. We will interpret it when we have the final gel tomorrow.

See you all tomorrow, when our next gel comes out at 11am 🙂

Good Evening Everyone Busy day today Myself Jo…

Amy Thompson / / Uncategorized

Good Evening Everyone!

Busy day today! Myself, Jo and Mike discussed possible modifications to the plug making method when using Propionibacterium acnes. We decided that we would need to use mutanolysin, which aids in cell lysis, as Propionibacterium acnes can be difficult to break into! We also decided to prolong the incubation time after the addition of mutanolysin and lysozyme from 2 hours to 3 hours at 37°C, in order to give them plenty to time to work on the Propionibacterium acnes.

We then began running through our first attempt at making the plugs! We started by adding chloramphenicol to our Propionibacterium acnes TYGE broth (that Mike inoculated for us). Once that had incubated for an hour we centrifuged it at full speed for 5mins, until we obtained a pellet. We then eluted this pellet in cell suspension buffer. We melted agarose and added these together. We then pipetted this into plug molds, which required a bit of practise! (we made 10 plugs) and left them at 4°C, to solidify. Once set we removed them into a universal tube and added lysozyme, it’s buffer and mutanolysin, it was then incubated at 37°C for 3 hours. We then removed the plugs and rinsed the plugs with pure water. We then added proteinase K and it’s buffer and left it to incubate at 50°C, until tomorrow morning.

We will be washing the plugs A LOT tomorrow! 4 times per plug 1 hour each, with wash buffer! We will then be working through the restriction enzyme digestion of the plugs info, and hopefully get ready to set up our first run with our hand made plugs! 🙂

See you all tomorrow!

Afternoon All Quite a quick day today Myself…

Amy Thompson / / Uncategorized

Afternoon All,

Quite a quick day today! Myself and Mike viewed the PFGE gel we ran last Thursday to Friday. Although the run appeared better in terms of visible band separations, the bands were still fairly faint and the wells appeared very dark. Mike said that this was due to too much DNA being left in the wells, we therefore decided to reduce the amount of plug we put into the well, by cutting the plugs as small as we could. We decided to reduce the buffer %, from 1% to 0.5%, to get rid of the initial 3 hour with 15 second switch time at 2.0 V/cm and add a second band marker (1Kb ladder). This 1Kb ladder was added as a reference in order to help us work out how many bases were present within samples.

We set up a PFGE run with all these new changes for 24 hours at 2.0 V/cm with a 45 second switch interval, and a second block for 1 hour at 0.6 V/cm with a 45 second switch interval in case we weren’t ready tomorrow morning to remove the gel. So we will see tomorrow what our changes have done! 🙂

Mike also showed me the TGYE agar he was making up, in order to re culture the Propionibacterium acnes samples. He showed me how to make up the agar, autoclave it and pour it. Tomorrow he is going to show me how to streak plate the Propionibacterium acnes samples onto the TGYE agar plates in the anaerobic cabinet. I have never used it before, so it should be really good to see!

See you all tomorrow! 🙂