Tag: 6919 strain

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.

Hey All Didn’t come in till late today…

Amy Thompson / / Uncategorized

Hey All!

Didn’t come in till late today, to allow Propionbacterium acnes 6919 strain sample to grow, however the sample hadn’t grown enough, so we have left it to grow till tomorrow.

Meanwhile, i removed the plugs from speI, washed them in 1x wash buffer for 30 mins, then incubated them in TAE 0.5x until i had made and set a new gel. I used the modified comb as before to create an extra large well, so i could use 2 plugs in 1 well, as the plugs are from an old batch and therefore still contain minimal DNA.

I then ran the PFGE for 15hrs, at 6V/cm, with 1-12sec switch intervals. Mike also suggested using an increased amount of buffer in a large container below, and pumping buffer through the PFGE to replace buffer as it left the PFGE back into the container. This will hopefully keep all the buffer in the container as cold as possible, reducing smearing and making bands clearer.

Tomorrow, if the cells have grown enough, i can start to make new plugs, with our modifications.

Hello Everyone First day back in the lab…

Amy Thompson / / Uncategorized

Hello Everyone!

First day back in the lab! Me and Mike had a quick chat about what i wanted to do next and made a plan for this week, and possible plans for my final few weeks in the lab – it’s gone soo fast!
Mike re cultured the Propionbacterium acnes 6919 strain samples to allow them all of today and tomorrow morning to grow. Meanwhile, i treated my 3 remaining plugs with restriction enzyme – SpeI, which needs to incubate overnight. I also prepared and autoclaved 3L of 0.5X TAE buffer ready for running a gel with the restriction enzyme treated plugs and the low range PFG marker, using the settings we decided on last time i was in the lab – 6V/cm, 15hrs, 1-12sec Switch Intervals and a 1.4% gel.

Tomorrow will be a late start, to give Propionbacterium acnes 6919 strain samples as much time as possible to grow, before i begin making new plugs with our decided on modifications and setting up and running a new gel with the restriction enzyme treated plugs and new settings. Hopefully we will get some clearer and just as positive results! 🙂

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! 🙂

Hello Filmed our newest videoblog this morning in…

Amy Thompson / / Uncategorized

Hello,

Filmed our newest videoblog this morning, in 5 mins! Our quickest yet – we are becoming pros! 🙂
Removed, stained and viewed our newest gel today. Unfortunately we got pretty conclusive results that our plugs were the problem. No separation was present from our old or new plugs, therefore next week we need to completely remake plugs.

We will be making 5 plugs all from Propionibacterium acnes 6919 strain, taking extra care to pipette very carefully, in order to produce better plugs. We will also only be producing our plugs up until treatment with lysozyme, mutanolysin and proteinase K, then running them on PFGE, to see if they are viable, before treating and running them with restriction enzyme, Spe1.

We are expecting to see one very clear band just below the wells, with no separations underneath and possible clear bands between the band and wells, as DNA should all still be intact and not multiple variable size fragments. If we see this, we will then treat the plugs with Spe1 and run them. We are expecting to see bands forming from the wells and towards the bottom of the gel, with clear bands throughout, but most dense around halfway down the gel, adjacent to our new low range PFG ladder, and the band – 100kb, as DNA will be lysed into approx. 86 fragments ranging from 817bp-13702bp, mostly around 10000bp (100kb).

Have a good weekend, and see you all on Monday 🙂

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 Everyone Came into the lab this morning…

Amy Thompson / / Uncategorized

Hey Everyone,

Came into the lab this morning to start to set up our next gel. I made up 3 L of TAE 0.5X, incubated 1 Propionibacterium acnes 6919 strain restriction enzyme plug and 1 non restriction enzyme plug in 1X wash buffer and then in 0.5X TAE buffer. I then made up a 1% agarose gel and left it to set. I put the gel and plugs into the fridge and left them until 5pm.

When i came in at 5pm, i cut and loaded 2 samples of Propionibacterium acnes 6919 strain restriction enzyme plug, 1 sample of non restriction enzyme plug and 1 NEB yeast ladder. I sealed the plugs and then ran the gel according to the info recommended on the low range PFG marker ladder, for 15hrs, at 6 V/cm, with a ramped switch interval from 1-12 seconds. I added a second block as the gel is only running for 15hrs, and will finish at 8.30am tomorrow morning, of 0.6V/cm for 2hrs with a 5 second switch interval.

We will hopefully have the new ladder tomorrow, so we can modify our new gel according to the results we get when the run finishes tomorrow and use the new ladder!

See you all tomorrow 🙂

Afternoon Everyone Today we started the looong washing…

Amy Thompson / / Uncategorized

Afternoon Everyone,

Today we started the looong washing phase of plug making! We washed all 24 plugs (2 per sample, and 4 per 6919 strain sample) as before, 4 times for an hour each in 1X wash buffer. We then separated off plugs from samples 2-11, added fresh 1X wash buffer and put them into the fridge for storage. We then washed the 4 6919 strain sample plugs with 0.1X wash buffer twice, and separated off 1 plug (not to be treated with restriction enzyme Spe1). We then added 1X Spe1 buffer to incubate the plugs, before removing this and adding fresh 1X Spe1 buffer, Spe1 enzyme and BSA. We then left the 3 plugs to incubate overnight at 37ºC.

While waiting for washes, we used online REviewer software to work out the number and size of fragments Spe1 will produce – 85 fragments ranging from 132702bp – 817bp, with the most fragments approx. 10000bp (100kb). We therefore decided to order BioLabs (NEB) low range PFG marker that ranges from 194000bp-2030bp (194-2.03kb), that will cover all the larger-medium size fragments only missing 4 smallest fragments. The image produced by REviewer is attached, showing all fragments along genome

Tomorrow, we will be setting up our next gel, using settings recommended by BioLabs (NEB) on the new ladder we have chosen to use. Hopefully we will have the new ladder very soon, so we can run it!

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! 🙂