Author: Deborah

Last day in the lab today Quite unexpectedly…

Deborah / / Uncategorized

Last day in the lab today 🙁
Quite unexpectedly the spread plates yesterday grew very well, over 100 colonies on each plate. I carried on to replica plate the ones with close to 100 colonies and incubated them over night at 37C. These also grew very well, i documented these down but i am not sure of what these results mean (if anything) as T0 did not yield enough growth to replica plate. Because it was just a plate count day i did pcr, using my hair, just as the results from before im a boy!?!

As i expected pALA1029 pOG04 DRW and pOG4003…

Deborah / / Uncategorized

As i expected, pALA1029, pOG04 DRW and pOG4003 have once again shown no growth, almost in sympathy the serial dilution i performed yesterday yielded poor results. The spread plates only showed colony growths of 1-14, as i was looking for colonies around 100, i decided that it was not worth replica plating. I have still carried out a serial dilution from the overnight broth cultures and spread plated them onto LB agar plates and left them to incubate at 37C overnight. I do not expect any growth based on todays results but one can hope!

Another frustrating day in the lab Grew pNC0412B…

Deborah / / Uncategorized

Another frustrating day in the lab!!
Grew pNC0412B to an OD of 1 then performed a serial dilution (A,B,C,D -1to-5.5), I innoculated a fresh 10ml LB broth with 100 microlitres from dilution -4 and placed into the 37C shaker overnight. I then spread plated 100 microlitres from dilution -5 onto a fresh LB agar plate, I also spread plated 100 microlitres from dilution -5.5 onto a fresh LB agar plate, then placed these into the 37C incubator overnight. I would have also done this for pOG04 NJC but it refused to grow, after 6 hours in the 37C shaker only an OD of 0.01 was achieved, i abandoned this set of broths. The plates i hoped to grow for today containing the plasmids pALA1029, pOG4003 and pOG04 DRW also showed no growth. I have tried again today so hopefully tomorrow will be a better day. I seriously think C2110 is the work of the devil!

Today may be Friday but because of the…

Deborah / / Uncategorized

Today may be Friday but because of the bank holiday monday, i am treating today like a monday!!
I am growing two batches of C2110 up to an OD of 0.35. Then i will perform cell competancy and transformation. Normally i would put the spread plates in the 37C incubator overnight but because of the bank holiday i am placing the plates in the 25C until tuesday. Hopefully there will be some growth. If all goes well Tuesday i will be working with pNC0412B, pOG04NJC, pALA1029 and pOG4003.

Had growth from two of my plasmids pOG04…

Deborah / / Uncategorized

Had growth from two of my plasmids, pOG04 DRW and pNC0412B but nothing from pOG04 NJC. I used an original sample of pNC0412B and so i am very happy that there is growth, this means that there is probably a fault with my plasmid prep. At least i have something to work with. The pOG04 NJC is a known temperemental plasmid so no growth is not wholly unexpected, just frustrating. Today just counted my T end plates and recorded the data, the rest of the day is spent making up agars and broths ready for next week as it is the last week in the lab and i would like to be prepared!

Hi all counted yesterdays plates and replica plated…

Deborah / / Uncategorized

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.

Today has been an almost perfect day in…

Deborah / / Uncategorized

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…

Deborah / / Uncategorized

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…

Deborah / / Uncategorized

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.

Hi all Catching up with last week the…

Deborah / / Uncategorized

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.

Groundhog day Today is still Monday no overnight…

Deborah / / Uncategorized

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.

Hey Yesterday i grew C2110 E coli to…

Deborah / / Uncategorized

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!!

Hey all Yesterday i took the overnight broths…

Deborah / / Uncategorized

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…

Deborah / / Uncategorized

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.

Really quiet day in the lab today was…

Deborah / / Uncategorized

Really quiet day in the lab, today was the first day of my Uros, plasmid stability experiment. Started the day by growing E.coli C2110 to an OD of 0.35, tough to guess when this happens, 0.38 was the final reading. Whilst waiting for the cells to grow i then innoculated another flask of LB broth with C2110 and placed it in the 37C shaker.The next task was to make the cells competant and then transform them, using the plasmids pOG04 NJC and pOG04 DRW. The final step was to innoculate an LB agar plate (containing Ampicillin 50) and incubate overnight at 37C.

Hey Today was a strange day Did my…

Deborah / / Uncategorized

Hey,
Today was a strange day, Did my final Tend plate count for the week. I inputted all of my weeks figures into a table and Nicola showed me some impressive maths to get to the percent stability of the plasmid used, in this case pOG 04. The percentage was 63, which was waaay out from the expected 3-5%. I am going to redo this plasmid next week and hopefully (wearing my lucky pants) everything will work out as expected. Did a little research into my eight final bacerial cultures still on my bench, i will not be defeated!!!
Also sad to see Sophie and Dan finish their project, both will be missed. Also going to be a little quiet without Amy and Jo whilst they’re n hols. Happy summer guys x

Hey Started the day by making 400ml of…

Deborah / / Uncategorized

Hey,
Started the day by making 400ml of LB agar and 200ml with AMP100 and 500ml of LB broth. Then i counted the T0 replica plates i did yesterday. Next i counted yesterdays T end plates and chose the ones with approx. 100 colonies and replica plated them onto LB agar and LB agar with AMP100. Then went to the presentation for poster presentation and project write up. Then i streaked a fresh LB agar plate with C2110 colony and then another LB agar plate with DH5alpha (for Rosie) and incubated them at 37C overnight, so that we have fresh colonies to work with next week. Nicola gave me my plasmids, popped them in the fridge. Nearly all prepped for next week. Also managed to make up stabs and slopes of E.coli e0791 that i brothed up yesterday. Also BIG thanks to Dan for his anthropology presentation and showing me the difference between boys and girls (on the skeletons). Very interesting seeing the ossicles and learning how to age and sex the bones.

Hi all Started day by completing my serial…

Deborah / / Uncategorized

Hi all,
Started day by completing my serial dilutions from yesterdays broth culture, I spread plated the dilutions of 10-6 and 10-6.5 on LB agar and incubated them overnight at 37C. Then taking the eight plates from yesterday I counted the colonies and chose the four with around 100 colonies. I then replica plated each of these onto a further two LB agar plates (one containing Ampicillin 100). These were then incubated at 37C overnight. I then also broth cultured another confirmed strain of E.coli (e0791).