Author: Amy Thompson

Evening Everyone 🙂

Today Me, Jo and Mike started our first run on the PGFE machine! We started by making up 2L of TAE buffer and our agarose gel. We used 3 standards: Lambda DNA and two types of yeast: one from BioRad and NEB. As we didn’t have our plug making comb, we used liquid lambda. We used BioRad yeast (Saccharomyces cerevisiae) that is a small strip of yeast embedded in agarose, that Mike then cut into strips, the size of a comb section. We also used NEB yeast (Saccharomyces cerevisiae) that is yeast embedded in agarose within a syringe, that Mike pushed out and cut into strips, the size of a comb section. Mike decided that this yeast would be the best to use for our project, due to it being easier to work with than the BioRad type.
We combined the Lambda DNA with pure water (from downstaires prep lab) and heated to 95°C, then cooled it slowly. Once cooled we added loading buffer. We also ran loading buffer alone, as a reference.
We attempted to start the cooling module, which should keep the PGFE at 14°C, but it wasn’t cooling the buffer properly, so Mike checked all the equipment, but couldn’t find any problems. He therefore decided to check with the manufacture this evening. Me and Jo also read through the manual for the PFGE and searched online for possible causes, with no luck.
In the meantime we decided to run our standards anyway, but pre cooled the buffer in the fridge and attempted to keep the temperature as low as possible. After Mike had cut the yeast plugs, he pushed them into the wells, we loaded the gel into the PGFE, added buffer, added the loading buffer into a final well, and began the PGFE run.
We then had a quick review of electrolysis, and discussed what gases were being released (H and O2), the size of these bubbles (H – small bubbles and O2 – large bubbles) and whether they were from anodes or cathodes (H – from cathode and O2 – from anode).

The PGFE is to run for 4 hrs at 2 V/cm, then for 14 hrs at 0.6 V/cm, and finish at 9am tomorrow morning!

So, we will see how it looks tomorrow! 🙂

Hey Everyone,

First proper day in the lab today! Me and Jo made up 1L of X50 TAE (a buffer which we will be using ALOT of during our project!) and 1L of EET (which is used when making plugs, we will be producing during the project, more stable), we then autoclaved both. Mike also gave us a reminder of our old friends; Moles and Molar Mass!
After the TAE was autoclaved we used it to run our first gel, in order to practise our technique. We diluted our TAE 50 fold and combined it with 8% agarose to make our gel. We pipetted out: 2 microlitres of sample, 2 microlitres of loading buffer and 6 TAE. 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 up our gel with ethidium bromide and used the UV spectrometer. Our gel turned out fairly well, although Mike said that the ladders were a little messy, and that could be due to the temperature. There was also a small nick in the gel, which created some dragging.

Excited for tomorrow! 🙂