"
Template:Team:UC Berkeley/Notebook/SC notes
From 2008.igem.org
Contents |
11 July 2008 (F)
This entire week has been quite a crazy week... and I suppose a notebook entry is long overdue. So, a (somewhat) brief recap:
Today:
Thursday (7/10):
Got there at 8:30 a.m. to run two gels from Madhvi/Cici's colony PCRs (22 samples total) to see if the actual PCR product size matched the predicted size. Then we started doing minipreps -- a LOT of minipreps. The original idea was to transfer all the growing colonies from 24 well blocks into individual 2mL tubes; but after deciding that it would be too great of a risk to do ~200 minipreps individually, we resorted to use a different method: all growing colonies would be placed in a 96-well block, miniprepped from same 96-well block with the use of a revised magnetic bead miniprep method. Hopefully, there would be less room for mess-ups. Towards the middle of the day, I miniprepped Cici's and my basic parts (since Cici wasn't here for the day), as well as 9 additional samples from a couple days ago. Having completed that, I aliquotted 8uL of each sample (just "colony 1") into 8 PCR tubes, and again, taped them to the wall outside the 3rd floor to be sent for sequencing. Our second round of basic part making is done! At the end of the day, I helped with re-growing the colonies that were miniprepped in individual 2mL tubes so that we can redo them on Friday using the magnetic bead method. And - to my surprise - I left at 6:45p.m.
Wednesday (7/9):
Got there first thing in the morning to run a gel on Madhvi/Cici's PCR samples from last night. Since Cici was not there in the morning, I picked the (two) colonies for each of our basic parts and grew them in media. I also discovered, while reading through my construction files, that my forward oligo for one of my basic parts was incorrect; it will have to be redone separately when the correct, re-ordered oligo arrives. Most of the rest of the day was basically devoted to picking colonies from our post-rescue (PR) stock plates to grow in 24 well blocks. Since there was a large number of them, it took quite sometime to do... We also had our modeling meeting in the late afternoon.
Tuesday (7/8):
Ran a gel for Cici's and my PCR samples from Monday for purification purposes; did digestion; clean-up with Zymo columns; ligation; and did rescue/transformation. Cici had to leave earlier that evening, so I completed the plating on my own. In between the process for making our basic parts, we helped out in a couple other tasks -- which may have included a purification(? - it has escaped my mind...). Since Cici left before she could start helping Madhvi with colony PCR, I stayed to do that for her. It took quite some time to find (in our documentation) the sizes of the PCR samples, so we could decide what program to run in the thermocycler... When that I was done, I stayed until 9:30 pm to help Bing pick colonies to grow.
7 July 2008 (M)
Today was a LONG and somewhat hectic day. I got there a little bit earlier to help with general preparation for the day: making sure we have enough pipette tips (we are in desperate need of p100 tips...!), restocking our toothpick arsenal, etc... In the earlier part of the morning 'til around noon, we helped with electronic organization on the assembly documents. I did a purification on 22 transfers that failed yesterday, but did not get to use a multipipetter, since we had so few samples to do...so, I did them one by one. That was quite an experience -- for my thumb, especially. Afterwards, I helped with more data inserting/organizing. Having completed that, Cici and I started working on our respective basic parts (the oligos had arrived last week, but we never had time to get to them). After making sure we knew which parts from our construction files actually had to be made, we got to work setting up for PCR. Cici, when finished, helped Madhvi with colony PCR and running gels on several transfer samples that were "questionable". There was a bit of confusion over some of the parts, when they realized that the actual parts, stock plates, and record documents did not always coincide! Meanwhile, I stayed to help with more data organizing; pick colonies (for parts that needed to be amplified) and grow them; and to watch part of a miniprep/purification with another set of transfers from the weekend. Cici and I were in the lab until 9:02 p.m. -- precisely.
3 July 2008 (Th)
The day was quite packed. The first task when I walked in the door was to help electronically record the "K" numbers of basic parts in the Lefty and Righty plates for transfer later in the day. Afterwards, I quasi helped with Digestion on the Lefty plate. 45 minutes after Digestion was completed, I set up for my assigned task of purification. We used Dr. Anderson’s magnetic plate, with one large magnetic sphere for every 4 wells. It was a little tricky at first to avoid removing the DNA, which was attached to the magnetic beads that attracted to the magnets; but we figured, that, by tilting the pipette tips to the sides of the wells away from the magnets, we could effectively remove the liquid while leaving most of the DNA at the sides. Once purification was done, I helped with Digestion on the Righty plate. While waiting for the Digestion reaction to occur, Terry discussed modeling with the team. Part of the way through, however, we had to stop and set up Righty plate purification. This second time doing the purification went more smoothly than with the Lefty plate. I finished purification at around 3:45pm, and decided that it was a good time for lunch!
The activity after lunch seemed a bit more scattered, but it was nonetheless interesting. First, I picked colonies for some of Madhvi’s samples and grew them in media. Immediately following that, I watched Jin do Purification with the 48 transfers from Wednesday. In this purification, we used beads that were coated with silica (?), and did not require the use of Isopropanol to precipitate the DNA. Then, there was an absolutely dreadful half-hour long lull of nothing to do. I was quite pleased when later, one - and only one - of my hands was requested to help with heat shocking (holding a couple samples in 42 C water). But, at least, one of my hands was useful for 90 seconds! Towards the end of the day, we plated the 32(?) heat shocked samples. Here, thankfully both hands came to good use!
2 July 2008 (W)
Today was a busier day.
A.M.: Finished construction files from yesterday.
P.M.: We started the assembly work today! After picking out 24 basic parts with C/A vectors and 24 with K/A vectors, I helped out with digestion; did purification; and watched ligation.
1 July 2008 (T)
Today: We continued to help with electronically sorting the basic parts later to be used in composite part building. In the earlier part of the afternoon, we picked colonies for the vectors that were being tested -- since we were desperately out of things to do. Towards evening, we were given the task of creating oligos and making construction files for a couple variations on existing basic parts. I was responsible for seven construction files: {rbs_barstar>}; {prepro>}, {a~prepro>}, and {rbs_prepro>} of both PhoA and LamB proteins.
Monday: There was not much for us to do during the morning. The vectors were still being tested to see whether they were functional, before we scale up and start doing actual assembly of composite parts. In the afternoon, however, we helped with electronically sorting out basic parts (with the C/A and A/C vectors) from the main list of basic parts. As for actual lab action: I learned to do the Agencourt SPRI cleanup on a couple of the vectors that were being tested.
28 June 2008 (Sa)
I wasn't working in the lab today; but, my sequencing results came back! All four of my tags have perfect reads.
27 June 2008 (F)
Today, we've completed one round of lab work from beginning to end -- from researching literature to sending out samples for sequencing! We finished doing minipreps in the morning, and (literally) taped our samples to the wall outside to be sent for sequencing. The results should come by tomorrow (Sat), so that we can analyze them! We also learned to run an analytical gel (using E-gel mode) on Molly's colony PCR to see if the products matched with the predicted size. At the end of the day, the team started planning out duties for everyone over the next couple weeks when we start building composite parts. Everything would be on a much larger scale; so the tactic is assign each person one specific job in the assembly line of jobs. As of present, I'll be handling the purification process (after digestion and before ligation).
26 June 2008 (Th)
It was a much lighter day today. I picked out two colonies to grow overnight tonight, and will miniprep them tomorrow. Since we had extra time on our hands, we also did a couple minipreps for Madhvi.
25 June 2008 (W)
Our busy schedule today:
We purified the PCR product (using Zymo cleanup); set up for restriction digest; cleaned up (Zymo) the digest; set up for Digestion; did Transformation; and, we learned how to use the "spreader method" to plate colonies -- without setting the ethanol on fire...
We finished at 8:15p.m.
24 June 2008 (T)
I finished creating/organizing my oligos and construction files onto spreadsheets and documents.
We did set up for the wobble PCRs; they're in the thermocycler overnight until tomorrow!
23 June 2008 (M)
Having completed one week of training/lectures, I've finally started on my minute portion of the lab work! Over last weekend, I searched for the DNA sequences of the FLAG-tag and the AP-tag. The objective was to find an existing E. coli vector for said sequences. Upon completing the search, I designed oligos for EIPCR for small parts under 30 bp (like our tags). When we discovered, however, that our sequences were too long - leaning precariously over the "30bp border" - we decided to prepare for a Wobble (overlap extension) PCR instead. This involved designing Fwd/Rev oligos that had a 20bp overlap. When that was done, we ordered the oligos. We now eagerly expect their arrival tomorrow at around 3p.m., when we can actually do the PCR reation!
