Tag: plasmid

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!

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 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.

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.

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.

Hi all Interesting day today the broths i…

Deborah / / Uncategorized

Hi all,
Interesting day today, the broths i innoculated friday were ready today so i made them into stabs and slopes. Finally my workspace is clearing, made up a few more bijou jars ready for the end of the week when i can hopefully add Enterobacter spp and Citrobacter spp to my completed list. I then went through the cell competency procedure with Nicola and she assaulted my brain with maths (hate maths mondays), we then went over plasmid stability and what we were to achive this week. in preparation we made up 400ml LB agar, 400ml LB agar with chl10, 200ml LB agar with Ampicillin 100 and 200ml LB agar with Ampicillin 50. Tomorrow serial dilution!