Tag: Videoblog

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 🙂

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 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! 🙂

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… 🙂

Hello Everyone Yesterday we ran a 24 hour…

Amy Thompson / / Uncategorized

Hello Everyone!

Yesterday we ran a 24 hour gel, at 4 V/cm with a 20 second switch interval PFGE gel. We used 3 large restriction enzyme treated plugs and a NEB ladder. Mike also asked us to research the genome for Propionibacterium acnes and the size of the fragments produced when lysed with SpeI. He asked us to do this so that we would be able to sketch a rough drawing of what we would really like to see on a perfect gel. Also to help us look for a more appropriate ladder to use with the Propionibacterium acnes samples, as the average size of a fragment is approx. 100kb, and the NEB yeast we are currently using runs from approx. 1900-225, not quite low enough to compare most fragments.

Today we observed the gel with Mike, which was how he expected it to look, with the NEB bands close together near the top of the gel. However, no bands were visible under the Propionibacterium acnes samples and Mike thinks we really need to have another go at making the plugs, perfecting the cell density and process. We also filmed our latest videoblog!

See you all on Monday 🙂

Hey All Not much to report today myself…

Amy Thompson / / Uncategorized

Hey All!

Not much to report today, myself and Jo got into the lab for 9am and tried to film our videoblog! Although someone couldn’t remember how to speak properly, so it took us atleast an hour to finally get some film we were really happy with 🙂

While we were filming we removed our gel, stained it and when we were finished viewed it under UV. We saved the image, as Mike wasn’t in today.

Third week done already! See you all on Monday 🙂

Evening Everyone Didn’t come into the lab till…

Amy Thompson / / Uncategorized

Evening Everyone!

Didn’t come into the lab till the afternoon today. Once we got in we removed the buffer from the plugs, and incubated them further in 1X wash buffer for 30 mins, at room temperature. While this was incubating myself and Jo made up 3L of 0.5X TAE buffer, melted agarose and then poured a new gel. The buffer was then removed and replaced with some of the 0.5X TAE buffer, that will be used to run the gel, to allow equilibration. We then cut and loaded 3 samples of fully treated Propionibacterium acnes DNA, 3 samples of Propionibacterium acnes DNA not treated with the restriction enzyme (SpeI) and 1 sample of lambda Hind III as a reference ladder.

Finally we sealed the plugs in with agarose and set the PFGE to run for: 12.7 hours at 6.0 V/cm with a ramping of 1-8 seconds, then 7 hours at 6.0 V/cm with a ramping of 0.1-2 seconds. We haven’t tried ramping before, but we decided to try this set up, as it is the method used in one of the journals we have been working from. Ramping is where one switch interval is used at the beginning of a time run, and another is used at the end, therefore over the time period the ramping slowly changes.

Tomorrow we will be filming our next videoblog, removing, staining and viewing our gel. As mike won’t be in, we will be discussing modifications and a plan for next week on Monday.

See you all tomorrow! 🙂

Afternoon all Just to let you know the…

Michael Shaw / / Uncategorized

Afternoon all…Just to let you know the hard work that Amy and Jo have been doing to VLOG (Video blog) as well as blog their progress is now live on Youtube. Both this blog and the vlog are new experiments for Nicki and myself to see just how useful/effective different means of web-sharing can be; maybe it’ll become something we embed into teaching from hereon in, maybe it won’t…only one way to find out.

UROS VLOG on Youtube

Afternoon Everyone Pretty short day today Myself and…

Amy Thompson / / Uncategorized

Afternoon Everyone!

Pretty short day today, Myself and Jo came in at 11am to film our latest videoblog (I’m starting to really enjoy making them!) and talk through our previously filmed material from last week with Mike. Our footage won’t be up for you all to view for a little while yet, but I will let you know when Mike makes it available!

We then removed our latest gel from the PFGE, stained it and viewed it. We have made some initial observations; that the separations unfortunately ran off the end of the gel and we also sized up the bands using the information within the NEB yeast package.

Myself and Jo decided on some modifications between us, and we will be reviewing these with Mike on Monday morning.

Can’t believe week 2 of the project is over already! See you all next week! 🙂

Hello Everyone Myself and Jo got into the…

Amy Thompson / / Uncategorized

Hello Everyone!

Myself and Jo got into the lab early this morning, in order to set up our next PFGE run with our new modifications. We made up 3L of 0.5X TAE buffer and 150ml of agarose (in order to reduce error), then left the gel to set. While it was setting we made up 50ml of agarose to seal the plugs into the wells. We then cut up 4 samples of NEB yeast, pushed them into the wells and sealed them up with agarose. We then set up the PFGE equipment to run at 6°C, for 15 hours at 6 V/cm with a 70 second switch interval and then for 11 hours at 6 V/cm with a 120 second switch interval.

The run is to finish at 12.15pm tomorrow, so myself and jo will be in the lab from 11am to film our second videoblog section! 🙂

See you all tomorrow morning!

Hey All Didn’t do very much today as…

Amy Thompson / / Uncategorized

Hey All! 🙂

Didn’t do very much today, as we are trying a 24 hour run with our new modifications we decided on yesterday. Jo and I came in at 9am, and made up 3L of TAE buffer, and left it to chill while we prepared our agarose gel. After we poured it and left it to set (which took a few goes today, as the gel box wasn’t tightened enough and gel kept escaping!) we prepared our samples. We used 2 repeats of BioRad yeast, 2 repeats of NEB yeast and loading buffer as a reference. For this run we also decided to seal the sample plugs within the wells with agarose, in order to promote the movement of the DNA from the wells and into the gel. We used a pipette tip cut with a knife, to give a larger, slanted tip, from which we pipetted agarose into the wells. This worked very well, and we managed to fill the wells easily. We then placed the gel into the PFGE, added buffer and our loading buffer. We turned on the pump to expel any bubbles trapped within the pump/tubes, and then ran the PFGE for 3 hours at 2.0 V/cm with a field switching time of 15 seconds. We decided to do this initial stage to give the DNA time to start moving into the gel, and hopefully allow clearer band separations.

At 2pm we returned to the lab to reprogramm the PFGE to run for 21 hours at 2.0 V/cm with a field switching time of 45 seconds, followed by 2 hours at 0.6 V/cm with a field switching time of 45 seconds. This run should finish at 11am tomorrow morning, therefore the second programm was put in incase we were not quite ready to remove the gel.

Tomorrow morning we will hopefully get to see how our modifications have improved our separations, and if not discuss further modifications for next week. We will also be filming our first video blog!

See you all tomorrow! 🙂