Tag: eppendorf tube

Hello All Unfortunately the re cultured samples hadn’t…

Amy Thompson / / Uncategorized

Hello All,

Unfortunately the re cultured samples hadn’t grown enough on Monday to use, so we left them another day and night until Tuesday. Today, although the growth was minimal we decided to start plug making. We added chloramphenicol to the samples, left them for an hour, and then centrifuged them all down until we had appropriate sized pellets. However, some of the samples did not readily form pellets, and several pellets were much smaller than others or were very dark in colour.

We then combined the pellets with cell suspension buffer, shook them to combine and added agarose. We then pipetted the samples into plug molds and left them to solidify in the fridge. Once solid, we pushed each plug into a separate eppendorf, added appropriate lysozyme, buffer and mutanolysin. However one plug hadn’t soldified enough and was discared. They are now incubating at 37C. At 6, i will remove the lysozyme, buffer and mutanolysin, and add proteinase K and buffer. Incubating them overnight at 50C.

Tomorrow we will be washing the plugs, and incubating them in restriction enzyme, buffer and BSA.

Evening Today we washed our plugs as before…

Amy Thompson / / Uncategorized

Evening,

Today we washed our plugs, as before in 1X wash buffer 4 times for an hour each. We then separated our 5 plugs into separate eppendorfs, and put 3 in storage in fresh 1X wash buffer. To the 2 remaining plugs we added 0.1X wash buffer for an hour.

To one we removed the wash buffer and added 1ml of Spe1 buffer to saturate for an hour. We then added fresh Spe1 buffer, Spe1 enzyme and BSA, and incubated overnight at 37C.

We set up a new gel in order to run the unrestriction treated plug, to ensure that the DNA within the samples are viable for use. If we see one large band just below the wells, that is as clear or clearer as our best previous gel run, then we will run a gel with the same settings, but with restriction enzyme treated plugs.

We made up 3L of TAE 0.5X and a 1% gel. We removed the wash buffer from the other plug and added 1ml of TAE 0.5X buffer to saturate it. Once the gel had set, we cut and placed 2 non restriction enzyme treated plug sections and 2 sections of the new low range PFG marker into the wells and sealed with agarose. We ran this gel at the low range PFG marker settings: 1% agarose gel, 15 hours, 1-12 second switch interval ramping and 6V/cm.

I also filmed with Sophia and Barney, regarding their experiences in the lab so far! 🙂

We will remove this gel tomorrow, stain it and view it. This will help us to decide whether to run the restriction enzyme plug, with the same low range PFG marker settings.

See you all tomorrow!

Evening All Didn’t come into the lab till…

Amy Thompson / / Uncategorized

Evening All,

Didn’t come into the lab till 4pm today, in order to allow our Propionibacterium acnes 6919 strain to grow sufficiently. We took 7ml of the Propionibacterium acnes 6919 strain added 14ul of chloramphenicol and incubated for an hour. Then we pipetted 1ml into 2 eppendorfs and spun them down for 5 mins at full speed. Once pellet, we added 250ul of cell suspension buffer into both, shook and then combined. We then added 500ul of melted agarose and pipetted into 5 plug molds. We then put them into the fridge to set.

We have left them in the fridge till tomorrow, when we will treat the plugs with lysozyme, mutanolysin and proteinase K.

See you all tomorrow! 🙂

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! 🙂

Evening all Today consisted of A LOT of…

Amy Thompson / / Uncategorized

Evening all!

Today consisted of A LOT of waiting! We started by washing the plugs in 1x wash buffer 4 times, for an hour each! This is to make sure that all the lysozyme and mutanolysin was removed. The plugs could then be stored in 1x wash buffer at 4°C.

We then put each plug into a separate eppendorf tube and washed once with 0.1x wash buffer for an hour, and then once more to ensure all EDTA had been removed, as it will remove cofactors that are very important to the restriction enzymes we want to use next! We then removed 3 plug eppendorfs, and put them into the fridge, in order to compare samples that were treated with restriction enzymes and those who weren’t. We removed the remaining buffer in the other 7, and added 1x restriction enzyme buffer to each, and left for an hour, at room temp. with gentle agitation. After this, we removed the restriction enzyme buffer, and added fresh restriction enzyme buffer, restriction enzyme (SpeI, which cuts at ACTAGT) and BSA (which acts as a crowder to increase conc. and allow increased collisions). This is to allow the fragmentation of our Propionibacterium acnes DNA, which is on average 100kb size fragments. We then incubated the plugs overnight.

Tomorrow we will be finishing off the plugs, preparing a new gel run and running a new gel with our home made plugs and new modifications.

See you all tomorrow! 🙂