Groovy GST

Not working again!

2006-05-25T13:49:00.000+01:00

No ransformation yet again. Running the ligation showed no bands on agarse gel, so likely using too low a concentration. This time i am using pretty much ALL my cut plasmid and a lot of my cut DNA and using no water in the ligation reaction - to increase concentration. It WILL work..... if it doesnt i am PCR original extract and then digesting that, will also be digesting more of my plasmid. Hopefully something will work.... it really needs to work or i will have no results!!

Finally!!

2006-05-24T11:34:00.000+01:00

Yesterday - just did transformation.... and read journals for intro

Today i checked on them and only positive control worked... again!! I thought about it and realised that i was using the same plasmid for ligation and acting as a positive control. The competent cells took up and were able to grow on kanomycin.... so that made me realise that i was using UNCUT plamsid for my ligation reactions.... no wonder they werent working!! Stoopid baka!! Ultimately i blame Princess, the cut plamsids were all on her microcentrifuge tube rack.... ok ok my incompetence played a small role.... fine a BIG role! Why am i so incompetent?!?! They SHOULD work now!! Tomorrow i can finally continue! (If it all goes well....)

Sleepy monday

2006-05-22T17:45:00.000+01:00

I slept at midnight and so really tired today for some weird reason.... need to sleep earlier (and ocne a upon a time 12 was reasonable) stoopid 9am starts. Practically wasted today (yet again) - i was suppose to do a ligation and then transformation using the EcoR1 digested inserts i prepared on friday..... somehow i threw them away..... i blame that on my purging of non-viable competent cells - my digested insert must have got mixed up with them... and in the bin it went. :'( SO had to re-do that..... ligation takes 4 hours (min.) and didnt manage to start it until 12:30, so didnt have time to do transformation which takes over 1 hour as it needs to be incubated..... yeah little achievements today.

Xi also visited, i am sure one of her blogs will give more details.

Friday again.....

2006-05-19T18:00:00.000+01:00

Intended to get in at 8:30 this morning.... it didnt happen!!!!! I got in at 9 started growth of my overnights to get that 0.5 OD600 reading, yet again. Needed to be finished at 12:30 to go to a seminal on fungi and nutrients - it was about networks, apaprently very good and put everything in Stumfs lectures into context. Yeah apparently - my stoopid fungi took longer than expected to grow. One culture grw by 11:45, the other i expected done before 12:15..... it didnt reach the right growth phase until 1!!! I did go down into the lecture, but it was pretty much over anyway - the question session was LONG! I thought i recognised Stumpf sitting in the row in front of me.... when he asked a question (his accent and the content - about protein interaction networks) i knew it was defiantly him!! Shame i missed it, was looking forward to it, and it shows the use of ISB!

The other thing i did today was restirction digest on my PCR product (yes again....). Monday it will be ligation and transformation. If that works then i will be one whole week behind, if it doesnt then i will be even further behind......... *sigh* REALLY REALY want it to work.

I got an E-mail from the library saying i have 3 overdue books - 2 PPEP and Lambs book..... i am SURE i brought them back......... oh well will check at home, if not there then i will have to talk to them.

Nightmare day

2006-05-18T17:40:00.000+01:00

When i entered the building Anna was already there and working (i was in shock, twice she has beaten me in!!).... she was talking to Derek.... i got good news immediately i had transformations although only positive controls. My ligated plasmids did not get taken up, so likely to be a problem with my ligation..... that needs repeating. Unfort it went downhill from there.....

Went to get ice, luckily there was still some left.... after Anna had been there! She took tons! She compressed it all nicely but trying to get an eppendorf tube in was a nightmare (glad i got my own). Throughout the day when i was getting my cultures out i heard a lot of cursing and angry inaudiable noises of people wanting ice... but none left!! Haha should have been there earlier.... My expression cells (B834) did not grow AGAIN!! I found out later that it was my fault, i added kanomycin when they do not have the resistance gene!!! So obviosuly there would be no governight growth! Duh! Also i somehow lost/misplaced my cloning cells so had to re-culture from Amelia again (my word she must be annoyed at that useless incompetenet 3rd yr student by now.....). I was growing the plasmid strain instead of the cloning strain, i grew that in LB overnight so they grew. Then i used LB+Tet and only the cloning strain has the tet resistance gene (its part of its genome apparently, i need to find out about it). So obviously throughout the day there was no growth..... again i ask where is my brain????

I also ran out of the PCRed DNA extract. So had to re-do them.... i ran 4 PCRs, checking on gel i only managed one! I was in shock! Even my beloved PCR went wrong!! It couldnt do this to me!!! Oh talking about ruunning gels, one of my demos used up the 1.2% agarose so i had to make some more..... i used the wrong agarose..... Derek didnt trust me with the microwave so he told me to let him do it! I felt such a little kid!!! He thought it looked weird, it was efficeinsing (bad spelling).... it was then that i found i used the wrong agarose... but apparently it shouldnt matter. After pouring it out the wells collapsed on themselves and they were just immpossible to run.... so i had to pour it into a beaker and then re-make it with the right agarose!! Using the wrong agarose resulted in softer gel.... Derek seemed to have fun playing with the gel in the beaker, he seemed fascianted that it felt so soft yet hard to break! That took far too long so in the end i didnt manage to do the ligation i wanted - oh well it doesnt matter i dont have competent cells to transform anyway.....

The demos got me a Victoria Sponge Cake to celebrate my b'day. Very nice and thoughtful. Yummy!!

Wasted day

2006-05-17T18:00:00.000+01:00

My transformations went wrong yet again!!! So i borrowed some more competent cells from Amelia (post-doc) and tried it yet again! Fun stuff! I was also suppose to make more competent cells but i didnt have enough CaCl₂ - it was frozen and when solid it looked enough.... but when defrosted well it wasnt nearly sufficient!! I didnt notice until late arvo and well the solution had to be autoclaved which takes three hours, so er yeah will have to do that again tomorrow instead, i will have to repeat it because the cells need to be in a certain growth phase meaning i basically wasted the whole day! Poo! Well on the plus side i did manage to start writing my methods, had a look at Mikes work and got a good idea how and what details to include. Muy useful.

Also apparently if the transformations i did today dont work then my super will have to stand behind me and watch to see what i am doing wrong.... scary stuff (and embrassing!!)

Summary: A wasted day. What seemed like a promosing project is going down the drain.

Long not so hard day

2006-05-16T15:31:00.000+01:00

The unfamiliarity is beggininng to disappear, i seem to know what i am doing (well perphaps its just an illusion seeing as this is the THIRD transformation i have attemtped, i am just hoping it will work out tomorrow). I used a PhD/post docs competent cells as well as my own "competent" cells. My own are non-viable (dead) they didnt even grow on LB without kanomycin..... it is likely to be due to me storing them at -20 degrees for a few days.... sigh. So i will have to make more competent cells tomorrow, so innoculated some specimen tubes with cloning and expression cells. If the transformation works tomorrow then i will only need expression cells. I should have been done around 1:30-2, but i was working so slowly that i didnt leave until 3 (not to mention helping Anna with her washing up), now at the comp room. Blogging and talking (Rakhel and Arunon) instead of working. But to be fair Arunon who also is doing transformations was asking me and literally testing me on why i was adding this or doing a certain step.... which could be useful in the viva.

Lab went wrong

2006-05-15T19:45:00.000+01:00

Over the weekend my transformations didnt grow - there was nothing on ANY of the plates!!!!!!! So i just re-did the transformations today using the same ligation left over from thursdays experiement. And i did another ligation, this time not doing any dilutions - there is something wrong with the spec machine, today when i re-spec my DNA it gave some minus figures 0_o. How can i have negative amounts of DNA?? SO that is the likely cause of not being able to get transformed cells. It was tested by running the original ligation reactions from thursday on agrose gel, indeed i had nothing! So how i expected my transformations to work with no plasmids i dont know! lol! So yeah that happened becasue the spec machine gave really high numbers, so consquently i didnt put much in, so in the end i ended up with no plasmid and no insert - so no ligation!

Tomorrow i am doing another transformation - this time with the products of the ligation reaction i am leaving in the fridge overnight. I threw away Steves spreader that i ruined.... Derek couldnt believe i was still trying to use that one! So he ended up making another, it was really cool!! I love how you can mould those glass pipettes! This time i read the protocol PROPERLY instead of skimming, and noticed the part i didnt read last time - sterilise using ethanol......... opps, no wonder! At least this time i didnt burn the spreader.......

I finished around 4:30, Anna was working for longer... all the demos/supers had to be out at 5 today.... so they kinda were rushing Rochelle and Anna, luckily they only had to do some innoculations, ok some might not be the word, they must have had 18 conical flasks between them!! (I didnt bother counting....) I was helping to move them to the 25 degree room. WE also bumped into Dr Lamb. And some of them had to go into the cold room, Anna was inside, and i was holding the door open, when suddenly anna shouts out "i beat you were planning on locking me in"!!! I had no such intention, in fact that didnt even cross my mind! All i wanted was to go home!! Lamb was smiling..... i dont even want to know what he thinks, probs something along the lines of "i cant believe they are final year students, so immature".

I also forgot my notepad today!!!!!!!!!! So will have to transfer all info from today into it. I also have a timetable planned, it looks rather relaxed... i also need to think of another biocide tp test, so far i have two. I will be doing iodide assays to test IPBC - like we did in EIM, and using HPLC to test the other! I think i will start to feel more relaxed when and if i ever get the final transformed cells (i need to get the plasmid from the cloning cell into the expression cell - so thats MORE transformations and plasmid preps, sigh!). Derek and i discussed what i wanted out of this project and where i wanted to take this project while we were sorting out my timetable. It was mainly transformation studies - taking the cells and overexpressing them seeing effects on biocide detoxication. He said he wasnt worried about my project, because i seemed to have good molecular tecniques.... i couldnt help it i burst out laughing! He seemed a bit shocked, i expalined that he was the first person to say that to me, he said "huh?" i was not sure whether i was mumbling or he didnt believe me, so i just said "usually my techniques arent so good" and left it at that! Its good to know my supers think i am good at lab.... We also discussed the literature review (aka intro) and i expressed my worry at the word limit 2000-3000 words..... but apparently my methods and results should be concise (me and concise 0_o), apparenly my results are mainly pictures and few sentences, my methods are usually well established protocols so dont need too much details. Thats good to know.

I was also told i didnt need to use the expensive white tips (the long thin ones) to load agrose gels!! But they are much cooler, and we ALWAYS used them in practicals.... oh well i will just have to stick with the yellow ones....

Why oh why?

2006-05-13T12:20:00.000+01:00

Yes i managed to get myself to start working in the morning, woke up around 10, started work 11ish. Did a good 30 mins before neighbour upstairs decides to play really loud Indian music! It has a really annoying solo humming tune, followed by a really fast and loud drum beat with Indian words.... he has played that song at least twice now, and few other songs. I can hear the stoopid song really clearly as if the music player was in my own room! Why are people so selfish? I dont want to listen to your stoopid music! Let me WORK! I WANT to work!

They keep stopping and starting, while the music is on i cant think let alone read! Then when music stops i get into reading my groovy GST journals... just get into it, then yeah that annoying tune again! Why oh why do they have to live upstairs!

Calm relaxing day

2006-05-12T15:11:00.000+01:00

Came in, defrosted my competent cells (which i had to move from the -20 freezer to -80 freezer). It took ages! It was suppose to stay cold, so had to leave it onto of ice and waited until like 11:30 for it to defrost. Derek stressed the importance of how fragile compentent cells are (they have their cell membranes lysed so they are able to take up plasmid). So i had trouble re-suspending them. They just werent re-suspending with the gentle finger flicks that the protocol recommended.... so i tilted the tube gently up and down..... i hope they are fine! I then transformed the competent cells - getting them to take up plasmid - it was just 5 mins ice, 30 second heat shock, 2 mins ice. Then incubate at 37 degrees for 1 hour... the shaker in the 37 degree room was not on, and there was a sticky label saying please do not turn on.... but super said whats the point in the shaker if its not on!! So turned it on.... i think someone just forgot to turn it on! How bizarre!

Then lunchtime! Unfort Anna, Marv and Chin werent ready. By the time they were i only had 20 mins left! Oh well the purpose of the incubation was to give the bacteria time to recover before subjecting them to another stressful condition - plating them on LB (Luria Bertani) and Kanomycin plates. I felt kind so let them recover for an extra 20 mins! Not that i was slacking! I decided to use the fume hood to plate my cells, no idea why! I just had my plates made on their so thought i would work there. Apparently it wasnt needed becasue nothing apart from my bacteria will grow on kanomycin media anyway.... but its good sterile technique! The gas for the bunsen is behind the fume hood! I had to get one of the supers to show me where it was! Its really hard to find.... well when you cant see where it is! No one was around when i finished, so i had to get on a chair to see and reach around!! I was using Steves spreader (it was made from a glass pipette - the ones we used in EIM to make capillary tubes) - it looked really artistic. I left it in the bunsen for a bit too long and it started drooping! My first instinct was to correct it with my fingers.... yeah i know stoopid! I burnt my fingers! And the spreader is slightly burnt...... and I completely ruined its rather artistic look. Oh dear.... Steve really wont like me by the end of FYP!! I did leave a note saying "Sorry Steve, i left it in the bunsen for too long and ruined your artwork", or something along those lines....

I stored all my competent cells in 1ml, but apparently its better to store in 1 microlitre samples - shorter defrost time, more convenient (seeing as thats the amount i use per transformation), and i wont have to keep defrosting and freezer. So defrosted the expression competent cells. Again that took ages! Well thats all i had to do.... leaving the plates over the weekend at room temperature. Finished before 3! Just in comp lab now... suppose to be looking for papers instead writing blog!

Bah long, waffly post full of jargon!

Hectic day

2006-05-11T18:42:00.000+01:00

Well came in at usual time today - 9am. Then it was striaght off to image my gel... er i left it over night and all the DNA had diffused out!! Apparently i should have stored the gel wrapped in cling film in the fridge! Oh well i will know next time! Gel ran fine, there was cut plasmid. Digested my PCR inserts and used alkaline phosphatase to remove phosphates from the vector so it wont religate (the insert did not get dephosphorylated therefore can get ligated into the plasmid - remember AMB!). Also because all our supervisors leave at 5 (they all get in REALLY early around 8ish) we have to leave at 5 as well..... I was allowed to run my gel until 5:30, Steve said he would wait, but i left to talk to marv and forgot about my gel, I think i was a few mins late, therefore Steve wasnt happy with me, and i got a ticking off. He had to be somewhere... he ended up leaving... opps!

Karen, Xiao and Marv went to Gloucester road for lunch, would have loved to join them BUT had to be back in labs early (30mins time). It was a really quick lunch without having to wait for EVERYONE to be ready....

After lunch it was more running gels, and then exising the flourescent bands using a UV machine and scrapel and then extracting the DNA from the gel. I had a hige panick when i speced my DNA and found there was no reading! Nothing. What happened to my DNA. I was thinking just great, i dont make any mistakes and it all works, BUT during the important parts i muck up! I would have to repeat all this weeks worth of lab work!!!! I was not a happy bunny. I was advised to re-spec them, however before i did, Derek noticed that i was NOT using the UV cuvettes! Lol, THATS why there was no reading! When i came to specing them i had WAY too much DNA, so i had to dilute them down, otherwise i would be adding 0.03microlitres! Too small an amount! I am leaving the cut plasmid and insert to ligate at 4 degrees in the fridge overnight. Apparently the enzyme works best in room temp. BUT becasue of too much kinetic energy in room temp the insert and plasmid dont stay long tog. to get ligated. The best method is to use low temp overnight or 16 degrees for 2 hrs (compromise). Left at 5:30. And went stright home without going to comp room!

Plasmid prep

2006-05-10T16:23:00.000+01:00

As each day goes on i seem to be plodding along at a slow and steady pace... it does not seem like i do much, there is a lot of waiting time. I cant seem to work in those short breaks either, by the time i get down to comp lab and start focusing on work, er its time to go back up again!

Summary of todays accomplishments: spin cells down, purified plasmid from cells and run on agrose gel; cut open the vectors with restriction enzymes (vector prep)and run on agrose gel. The rest will be frozen ready for ligating in my GST PCR products tomorrow.

I dont think i will be out of lab until 5:30 still need to wait 10 more mins for vector prep and then run gel (30 mins).

Its now official - the title of my FYP is:Construction of a glutathione-S-transferase expression vector and determination of its effect on biocide degradation
Second marker: Ramesh Wigneshwereraj (Biology). No idea who he is, just hoping he isnt too harsh!

Yay went right!

2006-05-09T17:34:00.000+01:00

Well went back up, tested OD₆₀₀, 0.2 something so waited for anna and had lunch with marv and chin.... i managed to leave my bacteria growing for an hour.... luckily for me when i rushed up the labs to test the OD it was at 0.530 perfect! What great luck! The other was still at 0.3xx so left them to grow some more..... harvested those at 0.479. Put them on ice and followed the procedure, managed to finish at 4:30. Yay! My only concern was that yesterday i used the centrifuge and today i was told i was meant to have used this other one, hope they both end up ok, i dont want to use another day repeating this really boring step!! THe other problem is i didnt put them on ice when i was re-suspening them, whats the problem? The bactiera MIGHT have been growing during the time that they were left out (luckily that was only for a few mins) and then they wont beat the right OD, so maybe left the growth phase.....

Dispite not achieving much today i am still happy for some reason, because it worked (i HOPE!)

Well i will be using them tomorrow, guess i will find out then, no point in worrying!

Not even 11am.........

2006-05-09T10:49:00.000+01:00

............ and i am already so confused! I am just repeating what i did yesterday.... have i told you about the loss of a free day? So tragic! Anyway i am so useless, i had no idea where the sterilised conical flasks were, so had to ask, they ended up being on the shelf to my right..... no idea how to use th autoclave, had to ask, no idea where the centrifuge tubes were, had to ask. I am sure the supers. must be sick and tired of me and my clueless, no idea where anything is, dont dont what i am doing self. It was so much easier yesterday when everything was already laid out! Oh well, the OD is currently at 0.112, need to check again now. I was also told that i didnt have to get it exactly on, around 0.5something or 0.7something will do, seeing as all i want is cells in the right phase..... so my 0.586 at OD₆₀₀ yesterday was fine!! Bah! I DID do it right!!!!! Boo!!

Oh well no use complaining now, i am in uni, and will just have to repeat the experiment. Oh well i should try and start that literature review on my next "break".

Stoopid Swipe card

2006-05-08T18:43:00.000+01:00

My swipe card is being stoopid! It will let me into labs, comp rooms, everywhere i want to go, but it wont let me print! It will beep at me everytime i swipe those stoopid panels on the printers!! Stoopid swipe card.

/Rant

My luck has finally ran out

2006-05-08T18:25:00.000+01:00

The day started off badly, i woke up late, so got into uni 20-30 mins late. Boo. And then from there it was downhill. I had to grow up E.coli (cloning and expression cultures) in LB and LB+Tet. It took forever to grow them up. I also had to get a OD600 of 0.5.... thats the difficult part! I managed ti get 0.522 and 0.526 for one of the cultures.... the other culture (expression) i overshot and the other failed to grow! Boo. IF this had worked i would have had a day off to do my literature review!!! Boo boo boo!!!!!!!!!!!! So i have to come in and repeat all this!!

The bacteria just didnt want to grow, i didnt have the right density (0.5 at OD600) until 5pm.... i still had seven other steps to do before i could go home!! I left at 6pm!! Boo!

The incompetenet at lab perevoso boy is finally showing himself.... i wondered how long my luck would last!!

Slow day

2006-05-05T19:46:00.000+01:00

Came in this morning at 9am, to start electrophesis of the PCR that i did yesterday. Continuing with my new found luck in labs they came out well! Then it was PCR again... and er wait for them to finish (three hours) before electrophoresis and imagine and then DNA purification. Thats all i did today! I also re-read two journals, my brain isnt absorbing, grr so have re-read some journals 3 times! Boo

Anna got violent with the conical flask using the magnetic stirrer and managed to break it! She seemed to have started a new trend, a whole lot of glassware was broken by one of the demos when he was looking for something on the shelves, and the other demo broke a big volumetric flask.... no idea how!

Well this weekend no plans, except work!! I want to write up my methods, start intro, search for more journals and have a rough idea about my aims/objectives and what i need to do next week ready for mondays lab meeting!

First day

2006-05-04T19:19:00.000+01:00

Anna agreeded to phone me when she got up this morning, i got up before my alarm clock, so turned it off (woke up at 7:30 0_o) re-read 2 lectures and then left for uni. Was early so sat around and skimmed another journal, waiting for anna to show up..... no anna so left at 9:30. Chat with Derek (super) before anna strolled in late. Discussed about projects then got showed A LOT of techniques, including PCR, DNA extraction, Gel electrophoresis (and how to make agrose gel), imaging the electrophoresis gel slab - stoopid comps wont let me use the program on my account, so had to get Derek to log in for me..... made LB broth (dont ask me to write what it stands for, i cant spell it!). Most of the techniques i used around twice. So a pretty packed day... although not as packed as Anna's! She didnt seem to stop working!! I couldnt believe that everything worked for me!! I can still remeber in AMB when we were purifying and extraxcting DNA, i got NOTHING, zilch, zero.... so i was pleasnatly surprised when it did work out! Derek was pleased too, apparently none of his previous FYP students had got the DNA extraction to work first time!! I was like 0_0 and speechless! I hope he doesnt get the wrong impression that i am good at lab work... becuase that can only end in disaster.

I like our lab, there are 2 phd students and Steve to help four of us, and they all seem to know what everyone is doing, they are keeping us organised and set us deadlines, our first is 25th May (i think, i have it written down in the cool notepad we were given to store all our info in!), and thats to finish the intro (what we are doing, why its important and all that jazz), it should be around 2000-3000 words apparently.... there goes my initial plan of writing intro by this weekend, instead aiming for 1000 words. Hopefully shouldnt be too bad. I also plan to write up what i did that day in the evening, and perphaps a results table. I also want to re-read some journals, Derek also wants an aims/objectives done so we can have a rough timetable drawn up. We will also be having weekly lab meetings to discuss what we will be aiming to do that week, so they can add their input etc.

A lot of work and i am shattered, but quite fun too! Bah, I was worrying needlessly last night... so didnt sleep too well last night. I suffered for it today, i felt my head wasnt screwed on, and i had trouble remebering what i was suppose to be doing, so anything i didnt jot down, well i forgot what i was suppose to add and how much! I was so paranoid that i had to check my basic arthimetic, 8x35....

Almost begininng

2006-05-02T23:58:00.000+01:00

The exams are over and we have a long extended weekend!! (my thoughts and all that jazz can be found on my main blog And it is almost time to start the final year projects (FYP). I will not start until thursday 4th May because out super is away until then.... instead i will spend the two days reading journals, 11 of them to be exact. Joy.

Meeting and beyond......

2006-02-27T23:00:00.000Z

After all the excitment has died down from finding out who has been allocated which project it comes to the series part - filling in forms and starting to think about what we are actually doing.

Had meeting with sup and PhD student who i will be working with. There was suppose to be four of us, but only Anna and I turned up... perphaps they didnt have 10:30 free, oh well. Well i have now got alot of reading to do - all later though. He said we only had to worry about it after exams. Goody! First week will be guided, after we can take the project in any direction we chose! Yeah! And unfort the directions we can take will be limited unless we get good results! Lol. Hopefully my technique isnt too bad. Techniques used? PCR, HPLC, and other molecular skills learnt in AMB i suppose. Will be making an expression vector - inserting GST into it. Using primer pairs somehow (i.e. i cant remeber what i will be doing with them!) Then cloning into E.coli. Afterwards changing expression to see what effects it has on the breakdown of biocides, including "compound X" (if you did EIM you know what this is!!), IPBC, and i can remember the other two!

New Blog

2006-02-27T22:48:00.000Z

Yes i decided to jump on the bandwagon and keep a journal about all things to do with FYP.

GSH (glutathione) is a thiol compound found in most aerobic organisms, it has many functions in the body including being an antioxidant and carb metabolism. GST (glutathione S-transferase) is an enzyme which catalyzes the conjugation of GSH sulfur atom to a range of different electophilic compounds including biocides which leads to its detoxification.

My project is taking the GST gene and inserting into a plasmid, then transforming bacteria with low/no endogenous GST production and not usually resistant to or able to degrade biocides including the broad spectrum fungicide chorothalonil.

My project will be between 4th May - 15th June 2006. My only concern is getting the gene into the plasmid, without that i wont be able to get very far with my project!! Well will keep you posted.