Tag: low range PFG marker

Hey Everyone Today unfortunatly the 10 different strain…

Amy Thompson / / Uncategorized

Hey Everyone,

Today, unfortunatly the 10 different strain samples of propionibacterium acnes hadn’t grown enough to use yet, so we left them to grow longer all today and overnight.

We then removed the previously incubated old batch of plugs from SpeI enzyme, buffer and BSA. We incubated them all in 1 x wash buffer for 30mins at R.T. During that time we made up 1L of TAE 0.5x buffer, autoclaved it, allowed it to cool and used it to saturate the plugs for 1hr. We also used the buffer to make up a 1.4% gel, before cutting each of the 10 different strain propionibacterium acnes plugs from an old batch and placing them into wells. We also included a sample of the low range PFG marker. We then sealed the plugs using 1.4% agarose, we found a few problems whilst doing this. As we haven’t worked with as many plugs in the same gel at once before, one pipette tip (1ml) wasn’t enough to do all 11 wells. This was also made worst by how quickly the agarose cooled in the pipette creating bubbling and messy sealing. We plan to use a different tip for each sample next time, and work with slightly warmer agarose.

We then ran this gel for 18hrs, at 6V/cm, with 1-12 sec Switch interval. We also used the modified buffer flow method – which took a few times to balance effectively.

Tomorrow we will be hopefully starting plug production and viewing this latest gel. See you all then 🙂

Hey All Today i came in and filmed…

Amy Thompson / / Uncategorized

Hey All,

Today i came in and filmed my latest videoblog. I then removed the plugs from speI, buffer and BSA, and added 0.1x wash buffer for 30mins. While this was incubating, i autoclaved 1L of 0.5x TAE buffer, which i then used to incubate the plugs for an hour. I then prepared a 1.4% gel, with modified comb to create a large well. Once this had solidified i added one plug of the low range PFG marker, one whole plug of restriction enzyme treated plug in the large well, and finally one 1/3 of a restriction enzyme treated plug in one well. I then sealed the plugs, and ran the gel at 6v/cm for 15hrs, with 1-12sec switch interval ramping.

Mike will be in tomorrow to removed the gel, stain it and view. He will then save an image for me to observe with him on Monday 🙂 See you all then!

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.

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! 🙂

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!

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 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!

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!