Not working again!

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!!

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

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.....

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

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

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

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.