Tag: PFGE

Hello Today was our last day in the…

Amy Thompson / / Uncategorized

Hello!

Today was our last day in the lab! 🙁 We filmed our latest and final videoblog, removed the gel, stained and viewed it. We saw some separation from all but one of the plugs, however separation was very blurred. We decided to run the same samples again, but for longer and with a higher switch interval. We set up and ran a new gel for 18 hours, at 6V/cm, with a 1-30 second switch interval, a 1.4% gel with modified buffer flow.
Mike will remove the gel tomorrow, stain it and view for us. Hopefully we will see better separation, from the samples containing enough DNA.

The last 10 weeks have been amazing – i have learnt so much since i started and it’s going to be really odd to not be in the lab everyday. I have really enjoyed working through a project, having to work on problems and figure out solutions. Then getting to see the good and bad results. I am very impressed with myself, as i set out to blog everyday i was in the lab with what i have done, the good and the bad! and i have actually managed it! 🙂 I wasn’t sure about whether i would get on with videoblogging at the beginning, but i have really enjoyed filming them every week – although most the content was never included as we ALWAYS got distracted! I really hope those who have watched them have found them as interesting and enjoyable as we did.

It has been a very rewarding experience, and i am so grateful to everyone who has made the last 10 weeks, the best summer – you all know who you are! And of course Mike, who has put up with us and our random chatter, better than i ever imagined he would. It has been an absolute pleasure to work along side him, and i hope i get the chance to do it again.

Thank you all for reading 🙂

Hey All Removed plugs from SpeI enzyme buffer…

Amy Thompson / / Uncategorized

Hey All,

Removed plugs from SpeI enzyme, buffer and BSA, and washed in 1x wash buffer for 30mins. We removed this, then incubated them in 0.5x TAE buffer for an hour, while we prepared a 1.4% agarose gel. We then loaded 1/2 of each plug strain into individual wells, as well as the low range PFG marker, sealed with agarose and ran at 6V/cm, for 15 hours, with a 1-12 sec switch interval, and modified buffer flow.

Tomorrow, we will remove the gel, stain it and view it. We will also be filming our last videoblog! 🙁 as it will be the last day of our project – 10 weeks has gone by sooo fast! We may also be running another gel, depending on the results of the first. See you all then 🙂

Hello Today we removed the plugs from proteinase…

Amy Thompson / / Uncategorized

Hello,

Today we removed the plugs from proteinase K, and washed them in 1 x wash buffer 4 times, for an hour each. We then incubated them in 0.1 x wash buffer for an hour, then in 1 x SpeI buffer to saturate for an hour. We then removed and added fresh SpeI buffer, SpeI enzyme and BSA and incubated overnight at 37C. We also made up 500ml of 0.5x TAE buffer and autoclaved it for tomorrow.

Unfortunately, we lost a few plugs along the way, due to instability. Tomorrow we will be incubating the plugs in 1 x wash buffer, then 0.5x TAE, before running the new plugs.

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.

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 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 🙂

Hello Today myself and Mike started by discussing…

Amy Thompson / / Uncategorized

Hello,

Today myself and Mike started by discussing the gel we ran last fri-sat. We produced pretty good results, as can be seen in the image attached. We could see much clearer bands, due to increased DNA, and slightly modified settings.

We therefore decided to start produced new plugs using the 10 different strains of Propionibacterium acnes we tried to use before. Mike re cultured these strains yesterday, therefore we left them for today to grow further, until tomorrow.

We decided to run the old plugs we produced using these 10 different strains, to confirm lack of DNA. We removed the buffer the plugs were in, added 1ml per plug of SpeI 1x wash buffer, to saturate for an hour at R.T. We then added 0.3ml SpeI buffer, 3ul BSA and 7.5ul SpeI enzyme to each plug and incubated overnight, and through tomorrow at 37C. We did have some issues with the SpeI enzyme, due to a lack of enzyme from our original batch, which was 50,000u/ml. The new SpeI enzyme was 10,000u/ml and therefore required some modified calculations, taking into account increased incubation time.

Tomorrow we will be checking the growth of the 10 different strains of Propionibacterium acnes and then beginning new plug making, if appropriate. We will also be removing the old plugs from SpeI enzyme, buffer and BSA, and washing them in 1x wash buffer before storing them till weds.

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!

Hey Everyone Today was a loooong day Came…

Amy Thompson / / Uncategorized

Hey Everyone,

Today was a loooong day! Came in at 9am, and as the plugs were still fairly cloudy, Mike suggested we leave them abit longer to allow further lysis, and therefore make the plugs clearer. We made up a fresh solution of lysozyme, proteinase K and mutanolysin, and left the plugs to incubate further until 12pm.

I then began washing the plugs in 1x wash buffer, 1hr each four times. Once finished i washed the plugs in 0.1x wash buffer for an hour twice. I then removed the wash buffer and added speI buffer to allow the plugs to incubate for an hour. After this i added fresh buffer, SpeI enzyme and BSA. The plugs will incubate overnight at 37C.

I made appropriate modifications in amount of lysozyme, mutanolysin, SpeI buffer and enzyme etc. In order to compensate for an increase in DNA conc.

Tomorrow i will be filming my new videoblog, then incubating the plugs in 1x wash buffer, and in 0.5x TAE buffer, before setting up and running a new gel with our new modifications, that looked like they worked very well on weds.

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! 🙂

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!

Afternoon Today myself and Jo removed the plugs…

Amy Thompson / / Uncategorized

Afternoon,

Today, myself and Jo removed the plugs from the molds into a universal, added lysozyme and mutanolysin and incubated the plugs for 4 hours at 37ºC. During that time we began filming interviews with other students in the lab, discussing their projects/work, positives, negatives and what they had enjoyed and found challenging. We then removed the lysozyme and mutanolsyin and added proteinase K, leaving the plugs to incubate overnight at 50ºC.

Tomorrow we will be washing the plugs, hopefully running a PFGE gel with non restriction enzyme plugs and treating other plugs with the restriction enzyme, Spe1. We will also hopefully be filming another interview in the lab! 🙂

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 🙂