Tag: Mike

Hello Today was our last day in the…

Amy Thompson / / Uncategorized

Hello!

Today was our last day in the lab! 🙁 We filmed our latest and final videoblog, removed the gel, stained and viewed it. We saw some separation from all but one of the plugs, however separation was very blurred. We decided to run the same samples again, but for longer and with a higher switch interval. We set up and ran a new gel for 18 hours, at 6V/cm, with a 1-30 second switch interval, a 1.4% gel with modified buffer flow.
Mike will remove the gel tomorrow, stain it and view for us. Hopefully we will see better separation, from the samples containing enough DNA.

The last 10 weeks have been amazing – i have learnt so much since i started and it’s going to be really odd to not be in the lab everyday. I have really enjoyed working through a project, having to work on problems and figure out solutions. Then getting to see the good and bad results. I am very impressed with myself, as i set out to blog everyday i was in the lab with what i have done, the good and the bad! and i have actually managed it! 🙂 I wasn’t sure about whether i would get on with videoblogging at the beginning, but i have really enjoyed filming them every week – although most the content was never included as we ALWAYS got distracted! I really hope those who have watched them have found them as interesting and enjoyable as we did.

It has been a very rewarding experience, and i am so grateful to everyone who has made the last 10 weeks, the best summer – you all know who you are! And of course Mike, who has put up with us and our random chatter, better than i ever imagined he would. It has been an absolute pleasure to work along side him, and i hope i get the chance to do it again.

Thank you all for reading 🙂

Hello Today we removed our latest gel stained…

Amy Thompson / / Uncategorized

Hello,

Today we removed our latest gel, stained it and viewed it. Unfortunately we didn’t get very good results – there was alot of blurring on one the low range PFG marker samples, lack of clear band separation on the low range PFG marker samples and no expected band just below the wells for the non restriction enzyme plugs.

We decided that this was most likely due to lack of cell density, and therefore lack of DNA which that could be seen on the gel. We discussed that this could most likely to be fixed by allowing the Propionbacterium acnes samples to grow longer and therefore increase the cell density and DNA, and possibly by centrifuging more of the sample, to reach a higher conc. We also re researched info on cell density within plugs in the manual and journals – we found that the optimum OD was around 0.8-1.0 and we had been working with 0.24. Because of this increase in cell density/conc. we will also need to increase our lysozyme, mutanolysin and proteinase K to compensate.

Mike asked us to run a standard gel, in order to see if there was actually any DNA present at all. We ran a 0.8% gel, for 2 and half hours at 120V/cm with a 1kb ladder. We saw very faint separation, that did confirm that DNA was present, but at very low density/conc. as expected. This also confirmed our modifications which we will put into practise when i come from holiday on Monday 6th August.

After speaking to Mike we decided to attempt to run a new PFGE gel using the whole Propionbacterium acnes restriction treated plug, in one well, in order to use as much DNA as possible, and hopefully see some separation showing multiple bands due to lysis. We ran this 1% gel at 6V/cm, for 15hours with 1-12 sec switch interval ramping, using a whole Propionbacterium acnes restriction treated plug and the low range PFG marker.

Tomorrow we will be viewing this gel and filming our latest videoblog! 🙂

Hi Started the day Gram staining E coli…

Deborah / / Uncategorized

Hi,
Started the day Gram staining E.coli a strain i dont have, this is another cultue confirmed so i then made a broth which i am incubating overnight at 37C. Spent most of the day waiting for cells to grow to an OD of 1, though 0.8 – 1.5 would be within range. Nicola went through serial dilution with me and showed me how to do the first set, i then completed the following three, broth culturing -4 dilution and spread plating -5 and -5.5 dilutions onto LB agar plates and incubated them overnight at 37C. Hopefully tomorrow we will have enough growth to count colonies (100 would be nice) then we can do replica plating. Had another Mike Shaw masterclass and debate (loving lab life xx).

Awesome day in the lab today Made up…

Deborah / / Uncategorized

Awesome day in the lab today,
Made up a broth of 1g Tryptone and 0.5g Soduim chloride in 100ml sterilised water. this was then dispensed into universal jars (4ml per universal) and autoclaved at 121C for 15 minutes. Using aseptic techniques i the innoculated the broths with Kl terrigena, Kl pneumoniae and Kl oxytoca these were incubated over night at 30C. So hopefully they will have enough growth so i can add Kovacks reagent tomorrow (indole test) and i can hopefully differentiate the three culures. I have confirmed Listeria monocytogenes and so have made a broth and will hopefully make up stabs and slopes tomorrow. Alice has kindley donated a pure sample of A. baumanni, which i streaked onto a nutrient agar plate and also innoculated a nutrient broth and incubated at 30C overnight. I have also confirmed Streptomyces aureofaciens which i will broth culture tomorrow. Mike was amazing and explained loading dyes in detail, and how pH can alter substances, and how dyes can be used to range DNA banding during electrophresis (bromphenyl blue around 300bp). Then mike sprinkled a red powder (flourescens) into a beaker of water, because of the pH change the water began to glow like a yellow highlighter pen. Mike explained why this happened (outer shell valence and refraction of light) but i was happy with my glowing water xx Next Nicola explained how she was to cut out DNA from the agarose gel she had just ran and the purpose of this. Then i watched as she cut out each band of target DNA so that tomorrow the agarose gel can be dissolved away to leave behind the desired DNA. I Iooked at the kit to do this and it is in no way as simple as it seems. Even had time for an ethics debate with Dan, Sophie, Rosie and Kamilla.