Tag: separation

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!

Afternoon Quite a quick day today came into…

Amy Thompson / / Uncategorized

Afternoon,

Quite a quick day today, came into the lab stained and viewed our gel. We saw little/no separation from the Propionibacterium acnes 6919 strain restriction enzyme plugs and the non restriction enzyme plugs. However we did see separation from the NEB yeast.

Myself and Jo decided that this may be due to our newest plugs. As mentioned before one of the pipettes we were using was full of liquid, and therefore may have given us incorrect amounts of substances when being used. We were unsure as to whether this was resulting in no separations from plugs on our latest gel, so we decided to run another gel with 2 samples of our old Propionibacterium acnes 6919 strain restriction enzyme plugs, and 2 samples of our new Propionibacterium acnes 6919 strain restriction enzyme plugs, along with one sample of new non restriction enzyme plug and 1 sample of NEB yeast.

We are hoping that by doing this we can establish if the lack of separation is due to our new plugs being poor, or the settings recommended by the new ladder – low range PFG marker, not being quite right. We set up the gel ad ran it with the same settings as yesterday – 15hrs, 6 V/cm, 1-12 second switch interval ramping, 1% agarose, and then a second block to hold the gel at 0.6 Vcm, 5 second switch interval until tomorrow morning when we will be in to remove it, stain it and view it.

Filming our newest videoblog tomorrow… 🙂

Hey All Today we came in removed stained…

Amy Thompson / / Uncategorized

Hey All,

Today we came in removed, stained and viewed our gel. The samples not treated with restriction enzyme showed a similar band, just underneath the wells. This was expected as the samples had not been treated and therefore lysed by the restriction enzymes, so separation according to size did not occur, producing only one/two bands. The samples treated with restriction enzyme showed some separation, but not enough, possibly due to not enough digestion. For both sample types it appeared that the largest plug size used worked the best. Mike also noted that this may be due to a lack of cell density within the plugs. The agarose gel also appeared very messy, and needed to be cleaned up. We destained the gel in TAE buffer, viewed it again and decided on the following modifications for our next PFGE run:

  • Run for 20 hours with a 20 second switch interval. The switch interval was decreased in order to produce better/clearer separation.
  • 3 large samples for each plug treated with restriction enzyme and those without, as the larger samples seemed to work the best.
  • We also deep cleaned our glass wear and autoclaved the buffer used within the sealing agarose, in order to try to clean up our gel as best as possible.
  • Finally, when we produce more plugs, we will try to increase the cell density within the plugs and modify our cell suspension buffer/centrifuge stage.
  • We are keeping the buffer, ladder, sealing in plugs, temperature and voltage the same.

Excited to see if our modifications produce clearer band separation 🙂

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! 🙂