Tag: SpeI

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.

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!

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.

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! 🙂

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!

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! 🙂

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 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! 🙂

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 🙂

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! 🙂

Evening all Today consisted of A LOT of…

Amy Thompson / / Uncategorized

Evening all!

Today consisted of A LOT of waiting! We started by washing the plugs in 1x wash buffer 4 times, for an hour each! This is to make sure that all the lysozyme and mutanolysin was removed. The plugs could then be stored in 1x wash buffer at 4°C.

We then put each plug into a separate eppendorf tube and washed once with 0.1x wash buffer for an hour, and then once more to ensure all EDTA had been removed, as it will remove cofactors that are very important to the restriction enzymes we want to use next! We then removed 3 plug eppendorfs, and put them into the fridge, in order to compare samples that were treated with restriction enzymes and those who weren’t. We removed the remaining buffer in the other 7, and added 1x restriction enzyme buffer to each, and left for an hour, at room temp. with gentle agitation. After this, we removed the restriction enzyme buffer, and added fresh restriction enzyme buffer, restriction enzyme (SpeI, which cuts at ACTAGT) and BSA (which acts as a crowder to increase conc. and allow increased collisions). This is to allow the fragmentation of our Propionibacterium acnes DNA, which is on average 100kb size fragments. We then incubated the plugs overnight.

Tomorrow we will be finishing off the plugs, preparing a new gel run and running a new gel with our home made plugs and new modifications.

See you all tomorrow! 🙂