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CAROL
Making Competent Cells
- Prepared sterilized LB broth for growing cells. Followed procedure found in the protocol page for making competent cells.
- After completion of making competent cells, transformed ligated products (pCS26+promoters ligated using NEB Quick Ligase) into XL Gold Ultracompetent cells. These cells were plated on LB+Kan agar plates and was incubated overnight at 37oC.
- As well, as a control, we transformed pBluescript into both XL Gold Ultracompetent cells and Top 10 Cells and plated cells on LB plates to incubate overnight at 37oC.
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CHINMOYEE
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EMILY
Verification Digest of J13002-LuxOD47E-B0015
- Did a miniprep of overnight cultures and took concentrations.
- Set up a Restriction Digest for verification (that the B0015 terminator is present in my contruct) with XbaI and PstI. If successful, we expected to see bands at about 1.7 kb and 3 kb for the contruct (J13002-LuxOD47E-B0015) and the psb1ac3 vector respectively.
Lane 1- J13002-LuxOD47E-B0015 C5, cut with XbaI and PstI
Lane 2- C5 uncut
Lane 3- C6 cut
Lane 4- C6 uncut
Lane 5- C7 cut
Lane 6- C7 uncut
Lane 7- C8 cut
Lane 8- C8 uncut
Lane 9- Size control- J13002-LuxOD47E
Lane 10- Size control- BBK LuxOD47E
- Analysis: From this gel it looks like colonies 5, 6 and 7 may have worked (contain the B0015 terminator) as we see two bands at the expected sizes. Colony 8 has some random bands, so I will ignore this one. Colony 6 looks the most promising as it is the cleanest (no random bands). I will proceed by sending this colony down for sequencing.
- Wrote out a lab blog for this week that I shall post tomorrow morning.
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FAHD
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IMAN
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JAMIE
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JEREMY
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KATIE
Implementing the Notecard Reader
The bacterial transformation activity was fixed in the morning and I added to the conditionals that tell you if the question has already been answered so once a question has been answered it cannot be answered again unless the quiz has been completed or reset at any time. I also added a further instructions option to the PCR machine, which gives a notecard for now, but I am interested in trying the instructions as audio. The instructions are for those who throw themselves into the lab first thing or those who need a reminder of how to operate their tools in second life.
The first and second construction site now use the notecard reader and I added more options to the Sequencing station so more can be sequenced. Now I just need to know the sequence of base pairs for each notecard I will be using. At the moment the notecards I am testing with contain random data. Ligating two genes together may be something I will be adding to the restriction digest activity once I can get it all working together since it is just adding more conditions. However, I would have to redesign how you could put things together so I believe I will hold off coding anymore of this until the activity has been finished. Tomorrow morning I can quickly complete the third construction site script and test with generic notecards.
One thing I'm not really comfortable with the dataserver event yet, which is very important to reading the notecard so I think it would be useful for me to trace the code within this event and determine exactly what it does just so I completely understand how notecard reading works.
Since we should probably get started on the base of the spiral that will contain levels I have started to write up some more notecards that are even more basic than the ones I have been writing (transcription, translation etc.) like:
- What is a gene?
- What is DNA? etc.
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KEVIN
1. Sequencing
I have sequenced by Colony 1 and 9 of my Pqrr4+Boo34 (RBS) because they were the closest to the positive controls and the cleanest lanes. For this, I have only used Biobrick CP forward sequencing primers, and no reverse. I only used forward and not reverse because the piece is quite short, and forward primer is enough to read every base pair. These are hopefully being sequenced right now and I am waiting for the result to be in.
2. Competent cells.
Two types of competent cells were made today, and they were TOP10 and XL Gold. Top 10 cells were made because we were running out on them, and XL gold was made because Top10 seemed to be not competent enough for Carol’s luciferase.
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MANDY
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PATRICK
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PRIMA
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STEFAN
Building inside the eukaryotic cell
I was working in Second Life for most of today, specifically the
eukaryotic cell. I scrapped the previous endoplasmic reticulum and
installed a new one and it looks much better. Scripts were added to
make it "sparkle" in order to give it that ribosomal look. Smooth ER
was also created today. Experimentation with particle systems proved
to be a worthwhile endeavor. I had trouble with changing the settings,
but I eventually figured out how. the result was vesicles coming out
of the Golgi body periodically (you should check it out!). I have also
chosen a location for the cell and thus, was able to script some of
the organelles to move and expand. Tomorrow there will be a meeting
with Gregor about ethics at 10 so that should be interesting.
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VICKI
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