Most summers we have around 20 students working in the Microbiology and Molecular Biology lab, SB209, in the Science Building on the Brayford Pool campus. This is a place for those students to share their experience.
//Today i counted my T0 plates and replica plated T end plates, I have tried again with pNC0412B, pOG04 NJC and pOG04 DRW. Hopefully tomorrow there will be growth as i have used the original plasmid preps. Time to finish up with the maths and percentages as there is only 1 week left to go!!
//Hi all,
counted yesterdays plates and replica plated pOG4 onto LB and LB containing amp100, then i counted and replica plated pnc0412B onto LB and LB containing chl20, then i incubated them overnight at 37C. My next task was to complete the second serial dilution and spread plated 10-5.5 and 10-6 onto LB agar plates with no antibiotics. Next i checked on pOG04NJC and pnc0412B – NO GROWTH!!! I am sure they know its monday! Having Checked with Ross williams he thinks it could be the plasmid at fault. Tomorrow i try again.
//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.
//Today has been an almost perfect day in the lab.
pOG4 and pNC0412B have grown quite nicely to an OD of 1 (0.8-1), I have performed a serial dilution on both and left the innoculated broths overnight in the shaker at 37C, the spread plates of 5 & 5.5 are in the 37C incubator. Also today i innoculated another flask of LB broth with C2110 and grew to an OD of 0.35. Then i carried out cell competancy then transformed them, using the plasmids pOG04 NJC and pNC0412B. I spread plated pOG04NJC onto an LB agar plate containing amp50 and spread plated pNC0412B onto an LB agar plated containing chl10. these were then placed in the 37C incubator overnight. Busy day tomorrow as agars, broths, velvets and NaCl need to be made-up/autoclaved, as well as tomorrows tasks. Early start i think!
//Hi all,
Counted the t end plates from yesterday and recorded the results, will spend the weekend doing some scary maths. My streak plate of C2110 grew nicely so i now have a fresh sample to work with on monday. And FINALLY i have managed to get some growth from pNC0412B so i have refridgerated the plate and will be working with the chloramphenical resistant plasmid on monday, pOG4 has shown a little growth so i have placed it in the 25C incubater. Hopefully enough will have grown by monday so that i can complete the whole set of plasmids i was given to test. Am really looking forward to monday.
//Hi,
Had a good couple of days, have been having trouble with growth – not yesterday each plate had 500+ colonies, think i need glasses now!! Today was an easy day too, counted yesterdays T end plates and recorded the results, then counted the plates from yesterdays serial dilution T0 plates and replica plated the ones with around 100 colonies, once again there was good growth so i replica plated ones with around 200 colonies.
//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 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!
//Hi all,
Catching up with last week, the plates i innoculated to try and trouble shoot the non growth issues i have been experiencing, showed that there is growth at each stage so at least i can rule out human error, yay! The strain of E.coli i am using (C2110) may be the issue as it is taking 6-7 hours to grow to an OD of 0.35, i would expect the growth to be in the region of 3 hours+. Leaving the plate with no growth on my desk to show mike the next day, miraculously i had fantastic growth overnight. I have already generated data for the plasmids pALA1029, pOG04 NJC, pOG04 DRW, pOG4003 and i am now starting with pOG4 and the Chloramphenicol resistant pNC0412B, fingers crossed all goes well.
//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 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 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 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.
//Groundhog day!!!!
Today is still Monday, no overnight growth again. Think C2110 is mocking me. I have repeated everything once again but i have tried to trouble shoot as to why nothing is working. Once i made thre cells competant i plated a sample onto LB agar plate and a sample onto LB agar with amp50. Then i continued on to the transformation stage, i then plated a sample onto LB agar plate and onto LB amp50 agar plate. I have also restreaked a fresh nutrient plate with C2110. Hopefully things will grow overnight, if not maybe i can work out at what stage things are going wrong.
//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, 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.
//Hey,
Yesterday i grew C2110 E. coli to an OD of 0.35. Three hours later!! I made the cells competant, then i transformed them, leaving them in the 37C shaker for 45 minutes. Once this was complete i spread plated 0.1ml onto an LB agar plate containing amp50 and left the plates to incubate overnight at 37C. Unfortunately this morning NOTHING had grown. very frustrating, so today is now monday and i’m repeating everything again!!
//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,
Yesterday i took the overnight broths for the two plasmids and completed another dilution series and spread plated the ones from -5.5 and -6. I incubated these at 37C overnight. Then i took out the T0 plates and counted the colonies, the ones that were around 100 i replica plated them onto an LB amp100 agar plate and then olnto an LB agar plate. These were then incubated overnight at 37C.
Today started out with me making more LB agar and LBamp100 agar plates, and making up 3x50ml LB broth, ready for monday morning. Then i autoclaved my velvets ready for next week. Once this was completed the science could start. I counted Tend plates and replica plated the ones with around 100 colonies, same as yesterday i replica plated 1x LBamp100 and 1xLB agar. The last job of the day was to count the colonies from yesterdays replica plating and record them. I also re streaked my C2110 so that i will be working with a fresh culture on monday.
//YAY!! got growth, finally can start. I took four colonies from each of my culture plates and made them up into broths using LB broth and adding ampiillin 100. Then i placed them in the shaker at 37C, until they grew to an OD of 1(0.8-1). then i set up a dilution series, labelling eppendorfs A,BC,D -1 to -5 adding 0.9ml NaCl and 0.684ml into the eppendorf marked -5.5. Once the broths reached an optical density of 1, 0.1ml was added to each eppendorfs with the exception of the -5.5 as 0.316ml broth was added. From the 10 -4 eppendorf, 100microlitres was innoculated into 10ml of LB broth and incubated overnight at 37C. The -5 and -5.5 dilution series were spread plated onto LB agar plates and incubated overnight at 37C. This was repeated for pOG04 NJC and pOG04 DRW plasmids. Cant wait for tomorrow.