Tag: centrifuged

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 All When we checked the growth of…

Amy Thompson / / Uncategorized

Hey All,

When we checked the growth of the 10 different strain samples of propionibacterium acnes this morning we were happy with most of them, expect a few which were still abit clear. We decided to proceed anyway with plug making, adding chloramphenicol to them for a hour, then centrifuging down 3ml of all samples.
We then centifuged the others further down until all pellets appeared the same size and roughly the same size as the previous most successful plug making. While doing this we melted agarose in a waterbath slowly at 50C.
Once all pellets were appropriate sizes, we added cell suspension buffer, eluted the pellets and added agarose. We kept the agarose, cell suspension buffer and cells at 50C, and pipetted each sample into a mold. Producing 1 plug per sample.
Once solidified, we pushed each plug into a separate universal, added lysozyme, buffer and mutanolysin. This is now incubating at 37C until half 7/half 8pm. After which Mike will remove lysozyme, buffer and mutanolysin and add proteinase K and buffer, incubating it at 50C until tomorrow morning.

Unfortunatly we ran out of lysozyme and proteinase K buffers, so Mike had us make up our own from 1M tris HCL pH8 buffer, sodium dodeyl sulphate (SDS), 0.5M EDTA pH8 and pure water.

Tomorrow we will be washing the plugs and incubating them in SpeI. See you all tomorrow!

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.

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! 🙂

Good Evening Everyone Busy day today Myself Jo…

Amy Thompson / / Uncategorized

Good Evening Everyone!

Busy day today! Myself, Jo and Mike discussed possible modifications to the plug making method when using Propionibacterium acnes. We decided that we would need to use mutanolysin, which aids in cell lysis, as Propionibacterium acnes can be difficult to break into! We also decided to prolong the incubation time after the addition of mutanolysin and lysozyme from 2 hours to 3 hours at 37°C, in order to give them plenty to time to work on the Propionibacterium acnes.

We then began running through our first attempt at making the plugs! We started by adding chloramphenicol to our Propionibacterium acnes TYGE broth (that Mike inoculated for us). Once that had incubated for an hour we centrifuged it at full speed for 5mins, until we obtained a pellet. We then eluted this pellet in cell suspension buffer. We melted agarose and added these together. We then pipetted this into plug molds, which required a bit of practise! (we made 10 plugs) and left them at 4°C, to solidify. Once set we removed them into a universal tube and added lysozyme, it’s buffer and mutanolysin, it was then incubated at 37°C for 3 hours. We then removed the plugs and rinsed the plugs with pure water. We then added proteinase K and it’s buffer and left it to incubate at 50°C, until tomorrow morning.

We will be washing the plugs A LOT tomorrow! 4 times per plug 1 hour each, with wash buffer! We will then be working through the restriction enzyme digestion of the plugs info, and hopefully get ready to set up our first run with our hand made plugs! 🙂

See you all tomorrow!