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.
//Hi all,
quiet day in the lab today. Made up 10X10ml universal bottles of MEA agar, as slopes so that tomorrow i can innoculate them with the confirmed funghi i have, Aspergillus paraciticus, Penicilin notartum and Aspergillus niger. Also made up 50ml of MEA broth so that i can hopefully encourage my remaining funghi to grow. Now to research Listeria
//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! 🙂
//Another long update post, must remember to do this more regularly!!!
So polywipes for our project still haven’t come in, but while we wait we are using spare supplies to see how long bacteria prevails on skeletal material. Unfortunately the results were a bit screwed up, so we have had to start again. On the bright side, despite the screwy results it looked like if there was anything on the bones it was gone after 24 hours, hopefully our repeat will show the same.
Also been very busy dealing with open days and work experiance students. On wednesday i think it was (Open day) we could barely do anything because of the prosepective new students and their families buzzing in and out asking questions. Had a workshop earlier last week as well, which was fun, teaching a class about how to build a biological profile from skeletal remains; some of the kids were really enthusiastic and eager, but as always there were a few who messed around and had me going up the wall!!!!!
Had a work experiance kid in with us for one day. His name was Luke, we showed him the bones and had him attempt to age, sex and calculate stature of one of the remains, and then showed him blood spatter and got our friend Hannah (Who is working with Dr Croxton on the composition of fingerprints) to talk about her project before we handed him back to Deborah for the afternoon, hope he had a good time.
All in all a busy and frustrating week, but i keep learning new tricks and tips and am still having a good time. Hopefully come wednesday i will have definitive results on the persistance of microbes on skeletal material (Just waiting overnight while the plates incubate) and possibly have our final supplies for the main project
//FAO: Sophia..the screen capture for finding primers for one of the aflatoxin genes in A.parasiticus
www.youtube.com/watch?v=dWAyyTKvcrw&feature=youtube_gdata_player
//Hi,
today Luke spent a little time with Sophie and Dan, Big thanks guys x. Then we made up few slides using Lactophenol cotton blue to verify the fungal cultures i have. Penicillin notatum, Aspergllus niger and Parasiticus,these are now ready for me to innoculate into MEA slopes next week. The only two fungi i have not been able to culture are Sacchromyces and Mucor but thanks to Mike i know where to begin on monday.
//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 🙂
//Taking this opportunity to share Mike’s blog at http://themolecularlab.tumblr.com/ which covers a lot of really relevant info on molecular biology, including PCR with details on primer design, and other areas. Critical reading for any students who are working on molecular biology projects.
//Hi,
today luke did water filtration with water from the Brayford and innoculated nutrient agar plates with the filtered discs and incubated them at 30C for two days. The mannitol salt agar plates that had been spread plated with water from the Brayford showed large yellowy mucoid colonies, this was a positive for a Staphylococcus species. Using a sample from the mannitol salt plates and from the E.coli luke streaked the day before, Sharon and Luke performed Gram stains to compare the colonies. We then made up 1000ml of 50X TAE, and a 1% agarose gel for Steve. I have confirmd two more of my bacterial cultures, hopefully tomorrow i can inoculate them into broths and then into stabs and slopes.
//Hi all,
firstly sharon, luke and I collected a water sample from the Brayford, then we each spread plated a couple of nutrient agar plates with 0.1ml of the Brayford water and incubated them at 30C for two days.Then Luke streaked a couple of nutrient agar plates with E.coli and incubated them overnight at 37C. Luke spread plated a couple of mannitol salt agar plates with the Brayford water. We then watched Rosie innoculate her plasmids with antibiotics as she explained to us what she hoped to find. Explained water filtration to Luke as this is our task for tomorrow.
//Hello Everyone!
Came into the lab today, removed, stained and viewed our latest gel. We observed a clear similar band under all the samples not treated with restriction enzyme, and slightly blurry similar band under all samples treated with restriction enzyme. Although the position of the bands are correct, the blurriness suggests that separation is still not occurring properly. There also appears to be a problem with the sealing agarose, resulting in very dark wells as DNA cannot properly leave the wells. This was actually my fault, as when making up the sealing agarose for the wells, i had been mixing the same 1g of agarose used in making the actual gel, in the smaller amount of buffer (50ml) used when making the sealing agarose, instead of halving the agarose to 0.5g. Therefore we were attempting to seal the wells with 2% agarose, which explains the difficulty we had in pipetting it (the formation of bubbles), the dark wells and smearing of samples through the gel.
We decided on the following modifications:
We then ran a standard 0.8% agarose gel, with 0.5X TAE buffer, for 2 hours at 100V. We ran 3 large samples of restriction enzyme treated plugs, 1 Kb ladder and lambda Hind III ladder. We sealed the plugs in with 0.8% agarose (0.4g agarose and 50ml 0.5X TAE buffer).
The results of this confirmed our modifications, and tomorrow we will be running a new PFGE gel for 24 hours, at 4 V/cm with a 20 second switch interval, applying our modifications.
See you all tomorrow 🙂
//Hi all,
Interesting day, started the day by making 3000ml of TAE buffer. Next i ran a PCR test with a sample of my hair. When we looked at the gel under the UV some showed two bands and some showed just one band, either i’m a boy or luke is a girl. Think we may need to redo the test and look up which banding type (1 or 2) shows a male or female. Researched a few of the bacterial cultures on my bench, some are same species where an indole test is the only way to differentiate them.
//Wonderful new website from Randall Monroe – creator of xkcd – proving that physics can be fun! www.what-if.xkcd.com
//Hey All,
Today we came in removed, stained and viewed our gel. The samples not treated with restriction enzyme showed a similar band, just underneath the wells. This was expected as the samples had not been treated and therefore lysed by the restriction enzymes, so separation according to size did not occur, producing only one/two bands. The samples treated with restriction enzyme showed some separation, but not enough, possibly due to not enough digestion. For both sample types it appeared that the largest plug size used worked the best. Mike also noted that this may be due to a lack of cell density within the plugs. The agarose gel also appeared very messy, and needed to be cleaned up. We destained the gel in TAE buffer, viewed it again and decided on the following modifications for our next PFGE run:
Excited to see if our modifications produce clearer band separation 🙂
//Hello,
Found 3 plasmids from 2 out of the 6 plates I did yesterday! So they have been placed into broth culture and tomorrow will be spread onto agar with different antibiotic discs to determine antibiotic resistance!
It workeedddddd! 🙂
//Hello!
Today i started producing competant cells and doing transformation.
Competent cells were made by first creating an LB broth by placing a colonie from a DH5 alpha plate into it, and having it incubated and shaking for around 5 hours! Once my cells were at the right optical density, I could proceed. As it happens they grew really fast and well so thats always a good start! Once I had centrifuged my cells down multiple times, placed them on ice, and added amounts of CaCl2 i ended up with cells which were then competent and capable of taking up plasmid DNA. The competent cells could be used immediately for transformation and (hopefully) uptake my plasmid DNA which i have obtained from my previous weeks in the lab!
I then began transformation where i added my different plasmid DNAs with some competant cells and proceeded to heat, cool, heat, cool etc! It is a very long process! But it enables the cells to recover and the antibiotic resistance of the plasmid to express itself 🙂
Basically with the solutions i then had, I spread 100ul from each one onto LB agar containing ampicillin, and if there are colonies present tomorrow morning then this means that the cells have uptaken my plasmids, and that i do have plasmids stocks that are resistant to amicillin! If i do i will have lots of things to play with 🙂 If not… i shall start all over again. 🙁
I have also spread a positive control, so that if it doesnt work tomorrow this will tell me if it was something in my methodology that was wrong or just that the plasmids in my DNA samples are not resistant to ampicillin, and it was probably just the chromosomes 🙁
Any questions feel free to come and ask! :):)
//Afternoon Everyone!
Started off today by viewing our latest gel with Mike. Unfortunately our samples ran completely off the gel, and therefore we only saw the areas where the samples had passed. We also saw that the wells were very dark, due to too much DNA still being present within them. We also used the wrong ladder for Propionibacterium acnes. Mike therefore decided that we should run our next gel for 24 hours, at 4V/cm with a 30 second switch interval time, based on our two best previous gels (PFGE 5 and 6). We also decided to cut our plugs into different sizes in order to see how the size of the plug would effect the gel. We cut the plugs into two small pieces, one medium piece and one large piece. We still used samples of both plugs treated with restriction enzyme and without. We also used a reference ladder of NEB yeast.
We then helped Mike to re culture the Propionibacterium acnes by making up TGYE agar, autoclaving it and pouring 15 plates 20ml each. Which i have seen done before, but never done myself, so it was good to have the opportunity to practise! Managed to pour them all pretty well 🙂
We came in at 5pm, to cut off the first well of the Propionibacterium acnes sample treated with restriction enzyme, to see it’s progress. We removed it, stained it and viewed it. We will interpret it when we have the final gel tomorrow.
See you all tomorrow, when our next gel comes out at 11am 🙂
//Hi all,
Checked Lactobacillus plates, Two do not look like the other three, after Gram staining a sample colony from all the plates, two had Gram positve small cocci. The other three showed long rod shaped gram negative chains, ths was more like what i expected to find. To be sure, i restreaked these onto M17agar with added Lactose and incubated them overnight at 37C. In preparation for tomorrows PCR tests i made up the solutions needed, working out quantities of HCl and Tris-HCl in 200mM. Microbiology is fun but maths makes my head hurt!!! big thanks to nicola for her little equation, i now can work molar solutions out but i think more practice is definately needed
//Hello!
So, as Dan has already mentioned, last week we spent a few days in the micro lab preparing nutrient agar plates to test our negative control of the polywipe sponges.
The polywipe sponges are out of date (Oct ’11) and therefore we needed to do a negative control to see if we could still use them to swab our bones.
We tested out 3 ways to put our sample onto the agar plate, a simple 0.1ml spread plate, another plate wiping the sponge itself onto the plate and the third way was by filtering the rest of the ringers solution onto a filter paper to place onto the agar. We learnt how to use the stomacher which is pretty simple so will be able to use that in the future for our project samples.
After incubating the plates at 37degrees for 24hours we could see some small colonies on the plates!! NOT WHAT WE WANTED! So then over the weekend we incubated them again this time at 25degrees to see if anything else grows.
Unfortunately it did 🙁 and now we can’t use the polywipe sponges and have to wait for new ones to be ordered in!!!
Today we checked up on our plates and recorded our observations, they seemed to be fungal growths on the plates so we sub-cultured them onto an MEA plate for curiosity as to where the fungi came from and possibly identify it. This has been incubated at 37degrees again and we will look at this tomorrow to see if anything has happened!
//Evening all x
Yesterday did a PCR test with my cheek cells, mouthwash was yuck! but good news is i’m human. Today was a day of filling in COSHH and risk assessment forms, definately NOT a fun part of my day, but necessary. Confirmed M.luteus so made up stabs and slopes, Chromobacterium CV026 also confirmed, made into stabs and slopes. Made up a batch of TAE and another litre of 50x (Tris base, glacial aceitic acid, EDTA and made up to 1L with distilled water). The main thing i learned, is to not go to lunch and leave the tris to dissolve. increased pressure made it near impossible to remove the stopper, fortunately we are scientists – heating the glass whilst running cold water on the lid, hey presto, the stopper comes off.
//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 🙂