Tag: DNA

Hello Everyone Not much to tell today our…

Amy Thompson / / Uncategorized

Hello Everyone,

Not much to tell today, our new plugs contained far too much DNA and cells, and therefore were very delicate and almost all broke apart during the removal of proteinase K, and addition of 1x wash buffer this morning. We decided to stop production of these plugs, and begin again next Monday as plug making takes around 3 whole days in a row. Hopefully we can perfect the amount of DNA and cells present in the new plugs, to avoid this problem, through measurement of OD/cell density after addition of cell suspension buffer after centifuge.

I will be carrying on writing my final report for the project for the next 2 days, instead of being in the lab and start fresh with lab work and plug making on Monday! See you all then 🙂

Hey Everyone Today was a loooong day Came…

Amy Thompson / / Uncategorized

Hey Everyone,

Today was a loooong day! Came in at 9am, and as the plugs were still fairly cloudy, Mike suggested we leave them abit longer to allow further lysis, and therefore make the plugs clearer. We made up a fresh solution of lysozyme, proteinase K and mutanolysin, and left the plugs to incubate further until 12pm.

I then began washing the plugs in 1x wash buffer, 1hr each four times. Once finished i washed the plugs in 0.1x wash buffer for an hour twice. I then removed the wash buffer and added speI buffer to allow the plugs to incubate for an hour. After this i added fresh buffer, SpeI enzyme and BSA. The plugs will incubate overnight at 37C.

I made appropriate modifications in amount of lysozyme, mutanolysin, SpeI buffer and enzyme etc. In order to compensate for an increase in DNA conc.

Tomorrow i will be filming my new videoblog, then incubating the plugs in 1x wash buffer, and in 0.5x TAE buffer, before setting up and running a new gel with our new modifications, that looked like they worked very well on weds.

Hello Today i began by removing the gel…

Amy Thompson / / Uncategorized

Hello,

Today i began by removing the gel, staining it and viewing it. The gel was much cleaner than before, and the low range PFG marker showed very clear bands, while the restriction enzyme treated plug showed some separation but blurring as before. This confirmed that the problem lies with the plugs, not the settings. Which will hopefully be cleaned up, after DNA conc. is increased in the new plugs.

I began plug making as the Propionbacterium acnes 6919 strain sample had nearly grown enough. Mike suggested i use 2 tubes of 10ml Propionbacterium acnes 6919 strain sample, add chloramphenicol and centrifuge down all 10ml of both samples. This produced a large pellet that i then eluted in cell suspension buffer and combined with agarose. I then pipetted this into 5 plugs molds, and left these to solidify. Once this had happened, i pushed them out into a universal, added lysozyme, buffer and mutanolysin and left to incubate at 37C, for 5 hrs. I then removed this, and added proteinase K and buffer, and incubated overnight at 50C.

Tomorrow i will begin washing the plugs, and treating them with SpeI.

Hey All Didn’t come in till late today…

Amy Thompson / / Uncategorized

Hey All!

Didn’t come in till late today, to allow Propionbacterium acnes 6919 strain sample to grow, however the sample hadn’t grown enough, so we have left it to grow till tomorrow.

Meanwhile, i removed the plugs from speI, washed them in 1x wash buffer for 30 mins, then incubated them in TAE 0.5x until i had made and set a new gel. I used the modified comb as before to create an extra large well, so i could use 2 plugs in 1 well, as the plugs are from an old batch and therefore still contain minimal DNA.

I then ran the PFGE for 15hrs, at 6V/cm, with 1-12sec switch intervals. Mike also suggested using an increased amount of buffer in a large container below, and pumping buffer through the PFGE to replace buffer as it left the PFGE back into the container. This will hopefully keep all the buffer in the container as cold as possible, reducing smearing and making bands clearer.

Tomorrow, if the cells have grown enough, i can start to make new plugs, with our modifications.

Hey all Today we removed stained and viewed…

Amy Thompson / / Uncategorized

Hey all!

Today we removed, stained and viewed our gel. We saw some very promising results! Although the separation wasn’t great, we saw a large blurred band near the bottom of the gel, near the middle of the low range marker bands present (which unfortunately were quite blurred and difficult to see). This confirmed the presence of DNA in the SpeI treated plug, if not very much, as estimated previously. However, enough DNA was present within the whole plug to show separation, confirming that our plug making procedure and settings were near enough on the right track!

Given the set backs we have been experiencing with the plugs this week and last, this was a real boost to get us excited to start putting our modifications into practise for plug making – increasing cell density/DNA and slightly modifying the PFGE settings – increasing the gel conc. from 1% to 1.4% in order to condense the ladder bands nearer the top, allowing further reference to the Propionbacterium acnes restriction enzyme treated plugs near the middle of the ladder.

We filmed our last videoblog for a week today, as i am away from the lab next week, this will also be my last blog for a week 🙁
Jo will also be away for the next 3 weeks, so we will not be able to put these new modifications into practise just yet! But i am very excited to get started, on what will hopefully be modifications that get us our best results yet for restriction enzyme treated and non restriction enzyme treated plugs on PFGE 🙂

See you all soon!

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! 🙂

Hello Everyone Came into the lab today removed…

Amy Thompson / / Uncategorized

Hello Everyone!

Came into the lab today, removed, stained and viewed our latest gel. We observed a clear similar band under all the samples not treated with restriction enzyme, and slightly blurry similar band under all samples treated with restriction enzyme. Although the position of the bands are correct, the blurriness suggests that separation is still not occurring properly. There also appears to be a problem with the sealing agarose, resulting in very dark wells as DNA cannot properly leave the wells. This was actually my fault, as when making up the sealing agarose for the wells, i had been mixing the same 1g of agarose used in making the actual gel, in the smaller amount of buffer (50ml) used when making the sealing agarose, instead of halving the agarose to 0.5g. Therefore we were attempting to seal the wells with 2% agarose, which explains the difficulty we had in pipetting it (the formation of bubbles), the dark wells and smearing of samples through the gel.

We decided on the following modifications:

  • Increase the agarose gel % from 1% to 1.3/1.4%, in order to help increase the separation of fragments.
  • Use the correct agarose gel % to seal the plugs into the wells, hopefully decreasing the dark wells and smearing, and increasing the movement of DNA through the gel clearly.

We then ran a standard 0.8% agarose gel, with 0.5X TAE buffer, for 2 hours at 100V. We ran 3 large samples of restriction enzyme treated plugs, 1 Kb ladder and lambda Hind III ladder. We sealed the plugs in with 0.8% agarose (0.4g agarose and 50ml 0.5X TAE buffer).

The results of this confirmed our modifications, and tomorrow we will be running a new PFGE gel for 24 hours, at 4 V/cm with a 20 second switch interval, applying our modifications.

See you all tomorrow 🙂

Hello Today i started producing competant cells and…

Rosie Myers / / Uncategorized

Hello!

Today i started producing competant cells and doing transformation.
Competent cells were made by first creating an LB broth by placing a colonie from a DH5 alpha plate into it, and having it incubated and shaking for around 5 hours! Once my cells were at the right optical density, I could proceed. As it happens they grew really fast and well so thats always a good start! Once I had centrifuged my cells down multiple times, placed them on ice, and added amounts of CaCl2 i ended up with cells which were then competent and capable of taking up plasmid DNA. The competent cells could be used immediately for transformation and (hopefully) uptake my plasmid DNA which i have obtained from my previous weeks in the lab!
I then began transformation where i added my different plasmid DNAs with some competant cells and proceeded to heat, cool, heat, cool etc! It is a very long process! But it enables the cells to recover and the antibiotic resistance of the plasmid to express itself 🙂
Basically with the solutions i then had, I spread 100ul from each one onto LB agar containing ampicillin, and if there are colonies present tomorrow morning then this means that the cells have uptaken my plasmids, and that i do have plasmids stocks that are resistant to amicillin! If i do i will have lots of things to play with 🙂 If not… i shall start all over again. 🙁
I have also spread a positive control, so that if it doesnt work tomorrow this will tell me if it was something in my methodology that was wrong or just that the plasmids in my DNA samples are not resistant to ampicillin, and it was probably just the chromosomes 🙁

Any questions feel free to come and ask! :):)

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 🙂

Afternoon Everyone Myself and Jo came in at…

Amy Thompson / / Uncategorized

Afternoon Everyone!

Myself and Jo came in at 9am this morning, and began filling out our COSHH and risk assessment forms. Although they were a bit of a challenge we managed to get them finished before our PFGE gel came out at 11.30am. Filling out the COSHH form involved making a list of all the chemicals we have used so far within our project, looking up their hazard category (using MDSS – which was very useful!), assessing their exposure potential (glacial acetic acid and ethidium bromide being our highest), steps we could take to protect ourselves (containment level), first aid and how to dispose of them. Our containment level for procedure was 1. The Risk assessment form involved identifying possible hazards within the lab, reviewing control measures we could take to reduce this risk and giving an overall risk assessment rating, ours was medium. I felt that filling out these forms was good practise as next year, during our final year projects, we will be required to do this ourselves for our own projects.

We also watched Mike re culture the Propionibacterium acnes in the anaerobic cabinet, which was really interesting to see!

We then removed, stained and viewed our gel. We managed to obtain much clearer bands and we no longer had very dark areas (large amounts of DNA) within the wells. However we did notice slight band elongation, which is caused by increased temperature. This is expected, as unfortunately the PFGE is still running at 25/26°C, instead of the 14°C it really should be running at.

We therefore decided to keep the smaller amount of DNA we were putting in the wells, keep sealing in the plugs, keep the buffer at 0.5X and not include the initial switch interval as before. In order to reduce the temperature and obtain clearer bands we decided to move the PFGE equipment into the fridge (to run at 6°C). In order to obtain more separation we decided to increase the voltage to 6.0 V/cm. We therefore set up and started a new run with these changes for 24 hours at 6.0 V/cm and with a 45 second switch interval, still using 2 samples of NEB yeast, 2 samples of BioRad yeast and 2 samples of 1Kb ladder. With a second block for 2 hours at 0.6 V/cm and with a 45 second switch interval, in case we weren’t ready to remove the gel.

Really excited to see how the massive change in temperature effects our separations! 🙂

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! 🙂