Tag: cell density

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! 🙂

Evening All Today myself and Jo started our…

Amy Thompson / / Uncategorized

Evening All,

Today myself and Jo started our second batch of plugs, made from 10 different Propionibacterium acnes patient samples and a Propionibacterium acnes 6919 strain. We followed the same protocol as before, but made 4 plugs from the Propionibacterium acnes 6919 strain and 2 plugs from each of the 10 different Propionibacterium acnes patient samples. Mike checked their OD to ensure appropriate cell density (0.25). We also centrifuged down 2 eppendorf tubes containing 1ml of the same sample to pellet, we then eluted the pellet in 200ul of suspension buffer each, in order to produce a cell concentration of 2 x 10^8 ,we then removed 100ul.

Having agarose divided into separate eppendorfs was very useful when melting agarose to combine with the centrifuged samples.
However we did experience a few problems, when trying to pipette the agarose and centrifuged sample into the molds some bubbles formed, and some samples appeared to contain less then others resulting in some plugs being half the size of others. We decided that this was most likely due to one of our pipettes being inaccurate due to liquid internally, and that we only produced exactly how much we needed, resulting in some being lost during the procedure. In order to correct this Mike took apart and emptied the pipette for us, and we planned to modify the method to produce slightly more (10%) to allow for losses.

After the plugs had set, we separated them all into separate eppendorf tubes, added lysozyme and buffer, mutanolysin and incubated for 3 hrs at 37ºC. We then added proteinase K and buffer and left overnight to incubate.

We also discussed more appropriate ladders with Mike, he showed us afew that might be better to use, such as http://www.neb.uk.com/productcatalogue/productinfotransfer.aspx?id=/New%20England%20Biolabs/Markers%20and%20Ladders/PFG%20Markers/N0340.
We decided that tomorrow during plug washing, we would use the online genome cutting software we have used before to find exactly how many and what size fragments Spe1 would produce when lysing Propionibacterium acnes so we could make a final decision on ladder size range, and order one asap.

See you all 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 🙂