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Hey all!
Today I have transformed some competent cells (DH5 alpha E.coli cells) and used four different plasmids pOG04, pOG4, pALA1029 and pOG4101 (pOG 4.003) and spread plated all the plasmids once they had been transformed and I have put them in a incubator overnight at 37 degrees! Tomorrow I am going to do alkaline lysis (plasmid DNA extraction) in the morning and then learn how to use restriction enzymes and do a restriction enzyme digest. I also plan to do gel electrophoresis with the different plasmids. I finally feel I’ve got the knack for using pipettes (A bit late I know) but i feel alot more confident with them.
Hope to see you all tomorrow! 🙂

Another great day in the lab. Completed stabs and slopes, now onto bacterial cultures i haven’t even heard of so monday is going to be fun. Understand a little more now why we use sybr green dye in electrophoresis and why it is sometimes better to use Ethidium bromide. Gram stained a known culture of E.coli using Lugol’s, will compare it on monday against a slide stained with Grams iodine, interested in what the difference is. Loving the research aspect of lab work.

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!

A rather sleepy blog today, waiting around in the lab for things to be ready is very tiring. With Rachael Simpson, we finished making our broth cultures into stabs and slopes and left them to incubate overnight. A slope is a universal jar filled (in this case) with nutrient agar and left to set at an angle, the target bacteria is innoculated onto the surface of the set, sloped agar, giving a larger surface area for the culture to form a bacterial lawn, these can then be stored for couple of months. A stab jar is a bijou jar also filled with nutrient agar, the target culture’s cells are stabbed a coulpe of times into the agar, then incubated overnight. The bijou jar can be stored for years. Both these procedures were carried out under aseptic techniques.

Hi, today we gram stained a few more cultures and confirmed what they were. We then made them into broth culture and incubated them at 37C overnight. Just pesky M.luteus to identify (which is rachael simpsons pet project). Learning so much about colony morphology. Also learned that not many people like to clear n clean their workspace when they leave!! working with bacteria, this is an important step/process.

Hi everyone, Faris and i started the day by making up 800ml of nutrient agar and 500ml of ringers. 2hr lunch whilst these autoclaved, 121c for 15minutes, but takes about an hour and half for the pressure to release and the temperature to drop below 80C. After lunch we poured the nutrient plates, then we made broth cultures and inubated them overnight at 37C, except B.cereus which is incubated at 30C. Then Rosie, Faris and I made agarose gels for use tomorrow. Had a great time watching Steve make up TAE buffer and making EDTA and learning how to pH it.

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! I need to get into the habit of documenting this every day so sorry for my lack of posting. I have made a big note on my whiteboard at home to post every day now! So i’ll fill you in what i’ve been doing with Nikki recently…
Last monday morning me and sophia made some LB agar (2 lots of 400ml) and made up four 100ml bottles of solution 1. In the afternoon once the LB agar had been autoclaved I made up 50 plates of LB agar and put 20mg of ampicillin in the LB agar and made up all the agar plates.
On Wednesday me and Nikki had a chat about what we’ll be doing and needs to be done for the project and I went home and did research on the project.

On Thursday I made up competent cells (using the DH5a E. coli cells) competent cells basically take up extracellular DNA… Anyway I had to grow the cells by using a colorimeter with the optical density at 600nm. The cells then had to be incubated to around 0.35. Unfortunately I left them in the incubator for a bit too long and they grew up to the optical density (at 600nm) of 0.41! (I’ve learnt that mistake early on now!)
Then I had to transform the cells (transformation is the alteration of a cell from the direct uptake of exogenous DNA from its surroundings and taken up through the cell membrane). i used four different main plasmids which were: pOG4, pOG04, pOG4101 and 1029. I then spread plated the different plasmids onto the LB agar that i’d made previously and left them to grow overnight in the incubator.

On the friday I then did plasmid DNA extraction on the DH5a cells and did alkaline lysis on the E. coli cells. (Alkaline lysis is a method used in molecular biology to isolate plasmid DNA from small volumes of bacteria!)

Today I met Rosie and she’s doing a similar project to me but with the brayford pool water. We worked together on the samples I’d extracted last week (doing the alkaline lysis) and did gel electrophoresis. We made up a 1% agarose gel into 100ml of TA bufer. 20 micro-litres needed to be used for the plasmid samples (of which there were 9 samples – Large pOG4A, pOG 4B, pOG 4 C, pOG 4 D, pOG 4 SUB, 1029 A, 1029 B, 1029 C and 1029 D) and 2 micro-litres needed to be used for the plasmids compared to when we did the DNA ladder of which 1micro-litre Was used but 20 micro-litres for both different types of samples. (The 20 micro-litres were made up 3 micro-litres of loading buffer, 2 micro-litres of sybur green and 14 micro-litres of water). The agarose gel was then put into the microwave to boil it and then had to be left to cool down and then was poured into the base.

Once the gel had set, the samples then went into the centrifuge for 5 seconds and then had to be put into the wells that had been made. There were 16 wells in total and two had to be left at each side. This then had to be left for an hour and a half for the proteins to move down. When the electrophoresis had been on for over an hour and a half; the gel was then put into the UV visulaiser. Mike showed me how to use it (which is pretty ace!) but unfortunately the sybur green didn’t work so we had to put the gel didn’t in ethidium bromide and left it for 10 minutes.
Once this had been left we then put it under the UV visulaiser again and found out that only the DNA ladders were showing up. It was then suggested to me that when I did the alkaline lysis method last week that I didn’t leave the pellets that were formed long enough to dry (Which I am still miffed at myself for!). So I’ve had to start from scratch.
I then made up 9 broth cultures using the Large pOG4A, pOG 4B, pOG 4 C, pOG 4 D, pOG 4 SUB, 1029 A, 1029 B, 1029 C and 1029 D again and have left them in a incubator over night at 37 degrees. I will be doing alkaline lysis and transformations tomorrow and I will be doing right this time! 😀