Tag: 1Kb ladder

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 🙂

Evening all Myself and Jo spent this morning…

Amy Thompson / / Uncategorized

Evening all!

Myself and Jo spent this morning researching the next stages of our project, then we came into the lab for 3pm, in order to take our gel out of the PFGE. We then stained it up and viewed it with Mike. We saw much better separation than before, with the bands reaching all the way to the bottom of the gel, and running off. Both yeast samples, BioRad and NEB, also appeared consistent with each other. The 1Kb ladder was not visible, which was expected. However, the separations slightly shifted to the right, producing wonky bands, that unfortunately weren’t very defined. Mike said this was most likely due to imperfections in the agarose, and we could rectify this by producing 150ml of agarose in order to reduce error. In order to try and stop the yeast sample running off the gel we decided to change our switch interval as well. We also decided that as the NEB yeast has been the most consistent and successful sample, we would stop running the BioRad yeast and 1Kb ladder. We will keep the size of the plugs the same, and well as sealing them with agarose.

Reviewing the NEB yeast information we decided to try running our next gel following the guidelines provided, 1% agarose, 15 hours at 6 V/cm with a 70 second switch interval and then 11 hours at 6 V/cm with a 120 second switch interval. Using 0.5X TAE buffer, 2 samples of NEB yeast and at 6°C as before.

See you all tomorrow morning as we set up our next, and possible last gel before starting on our next stage in the project! 🙂

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!