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31 July 2008 (Th)
This morning, I checked my plate for transfer sca9, and it looked quite unpromising. There were only two colonies growing on the plate. I picked them anyway, streaked them on Spec and CA plate, and grew them in CA media. Miniprepps tomorrow!

Cici miniprepped the sca5 and sca11 (HA tags). They digestion mapped well, and sca5-2 and sca11-1 were sent out for sequencing today.

Re-sequencing results for sca2, 3 and 4 also came back today. Sca2 again, did not turn out right; while sca3 and sca4 are being re-amplified so they could sequence it again. Jin said to re-transfer it. So, more waiting ensues, I suppose.

Aside from that, I did a couple miniprepps for Bing.

Today, I tried the new protocol for transfers that Dr. Anderson suggested on sca2:

New Transfers Method:

Digestion: 0.5ul plasmid in entry vector 0.5ul EcoRI 0.5ul BamHI 1ul NEB2 + ATP buffer 7.5ul of water incubate the digestion for 1hr. in the thermocycler, and 20 min for heat kill. No purification step. Ligation: 1ul of dilute assembly vector (or 0.5ul if undiluted) 0.5ul ligase Transform. (same as usual)

Hopefully it works better than our previous transfer method.

30 July 2008 (W)
Sequencing results came in at 11:48 a.m. - exactly. (Many of us were sitting at the edge of our chairs, just dying to know how our samples fared.) As happy as I was that results for our 9 samples came back, my happiness vanished like the morning mist (boredom lends much more room for descriptive writing and similes...) when I found out that four of our samples did not turn out right. Sca2, 3, 4 came out with bad reads; they're being resequenced today. Sca9 came out as a good read, but it turned out to just be some random sequence, not in anyway ressembling the {<phoA!} we were expecting. Even though 4 out of 9 samples were problematic, we still could not start assembly today: all of our planned assemblies had either one or both of the problematic transfers in them! Oh, the rage!!!!

The only thing left to do today is to retransfer (from stock plate 3.2) sca9, which we know for sure is incorrect.

29 July 2008 (T)
The prepros that were sequenced yesterday came out fine. They came out to be perfect reads.

On a much more dramatic note, we realized today that all 9 transfers that I thought I had sent for sequencing never actually made it to the wall on Friday! This morning, when Cici was looking for her beloved pink rack, we found our sequencing samples, sitting there ever so comfortably and innocently, exactly where I had left them -- just waiting to be taped to the sequencing wall!!! Before I left on Friday, I was pretty certain that I at least let somebody (whoever that may be) know that my 9 transfers needed to be taken upstairs. Apparently, in the rush to get all sequencing samples up to the 3rd floor by 6:45p.m., those poor little samples were forgotten! At any rate, the important and sad truth was that none of our samples made it to the wall on Friday!!!! I personally took them upstairs this morning and taped it to the wall. Results -- oh, long-awaited results! -- should come in by tomorrow morning. Besides that, I helped Jin with Zymo for 7 samples: 6 of which were assembly vectors, and 1 of which is a mystery to me.

Hopefully, I'll have something more to do tomorrow when sequencing results come back.

28 July 2008 (M)
After a restful weekend, I came back this morning to finish miniprepping my prepro samples that Jin finished for me on Friday evening, and for which Bing picked 2 colonies each. Besides that, I organized more spreadsheets; restacked/reorganized the ever-growing, ever-towering stacks of agar plates in the fridge; and helped do a digestion map. It seems that running gels and organizing things have become our newest, favorite pastime --- it makes the waiting for sequencing results less..."on edge".

Other than that, it was just good to be back at the lab...

25 July 2008 (F)
Came in this morning to do some electronic organization (yay spreadsheets!)

This morning, I checked the plates and tubes in which our transfers grew. Most of the tubes had a decent amount of culture in there; however, 2 of the tubes (sca8 and sca14) did not seem to have much growth. After letting Jin take a look, we decided to let the tubes sit in the warm room for an extended period of time, hopefully so that more colonies could grow.

For most of the morning, there was not much lab work that I personally had to do. Besides organizing spreadsheets, and scribbling, and re-scribbling notes and schedules, I helped Bing prepare digestion for 8 of his samples. While those were sitting for an hour digestion, I helped Jin add 100ul of ADB buffer to four of his samples. After having lunch as a collective group, I helped Bing run a digestion map on his samples.

At 3 p.m., it started to get really busy: our 12 tubes of transfers were ready to be miniprepped -- Cici took care of that.Then, Jin came back with the new oligos for the parts that had to be re-done. I set up PCR for my 6 prepro sequences, as well as Cici's  and <HA! tags. The tags were done in the thermocycler really soon, so I zymo purified them, along with 2 of Bing's samples. Then, I digested those 2 tags, and set them in the incubator for one hour. Then, I helped make a digestion mastermix for 32 samples (our 12 transfers, and Bing's 20 assembly samples). I aliquoted the mastermix, and Bing added the respective miniprepped DNA into each tubule. Those also sat in the incubator for around 45 minutes. Towards 6:30pm, we were racing the clock to digestion map said 32 samples, and get them up to the 3rd floor wall to be sent off for sequencing. While the digestion mapping was happening, I labeled tubes and aliquotted the respective DNA samples into the tubes to be sent for sequencing.

After leaving Cici with a couple final instructions for finishing the tags, and knowing that my prepros would be in good hands, I decidedly left the lab at 7:15p.m.

24 July 2008 (Th)
I came in at 9 a.m. to check assemblies sca15 and sca21 and their co-transformation plates; also checked our PR plate fimH>'s. All 12 colonies I picked for sca21 co-transformed; 2 of 12 colonies of sca15 co-transformed; and 2 colonies of PR fimH>'s out of 5 grew.

Miniprepped sca15-1, sca15-2, sca15-3; sca3(fimH>)-2, sca3-4.

10 a.m.: minimeeting

Digestion mapped all 5 samples, all of which came out fine. Sent sca15-1 and sca3-2 for sequencing.

At the minimeeting, we decided to assemble with the 1-2-3 method, using less plasmid DNA. Results should hopefully be "cleaner", and there should be a lower co-transformation rate. However, since none of our transfers were in their respective Lefty (BglII methylated) and Righty (BamHI methylated) cells, we had to transfer into said Lefty and Righty cells.

List of transfers:

name     antiB     cell sca1        AC       L sca2         CK       R (sca3)       KA       L sca4         AC       R sca6         AC       R sca7         AC       R sca8         AC       R sca9         CA       L sca14        AK       R

The transfer protocol we used was a little bit different, the most exciting part of it being that we could directly put the cells into media without having to first wait for the colonies to grow on the plate:

TRANSFERS FROM MINIPREPPED DNA (ALREADY IN CORRECT VECTORS) (transfers into L/R cells) In 0.5 ml tube, combine: Take the reaction, and: Then, incubate your tubes for 5 minutes (No rescuing is necessary) -- Prepare plates (double antibiotics) and tubes with media (LB + antibiotics) Take 15ul of rxn. Spot it onto a plate (no spreading required) Take the remaining 15ul into a pipette tip. Drop the entire tip into your tube of media. Let plates and tubes sit overnight.
 * 1ul plasmid dna
 * 30ul of cells (L/R) + 30 KCM (no water)
 * sit in ice for 10 min
 * heat shock for 90 sec
 * sit another 2 min ice

Miniprep the NEXT DAY!!!!

The plates and tubes are incubating overnight.

Additionally, Jin discovered that some of the basic parts (2 tags, and prepro sequences) were wrong -- they had extra base pairs, creating a potential for frameshift!  and  sample (from 7/3 Stock Plate #4) that grew up last night. We digestion mapped it, but unfortunately, it was faulty. It will be another day-long delay for all our Assemblies that need {fimH>}!! Jin suggested that we sequence it anyway, just to see what happened. Then, onto plan B: 1.)pick 5 colonies of fimH> from the post rescue plates (#6, Well B12), and streak on KA and spec plates to check for coT; and simultaneously, 2.) Take fimH> miniprepped DNA (from 3.2 stock plate, Well E2), and do a transfer with it (into AK vector). Hopefully, method 1 will yield fimH> that we can use by tomorrow to do assembly. Basically, I handled all of method 2. Though it was time consuming, the positive outlook is that I now have one complete transfer process under my belt.

The other "major" task we did today was re-assemble sca15 and sca21. Cici was feeling uncomfortable about the assembly we did for sca15 and sca21, because one of the tubes was labeled incorrectly...and she was worried that the parts were not assembled correctly.

Finally, I picked colonies for sca15 and sca21 that was done yesterday. We now incubate the plates for a longer period of time for optimal bacteria growth. It should be interesting to see if this new incubation method alters the co-transformation rate....

22 July 2008 (T)
I came in today to find all 10 colonies of sca21 co-transformed...which means that we have to re-assemble it also! It also turns out that fimH> never got amplified overnight, so that had to be redone again as well. Until fimH> is amplified, there are not many assemblies that we could redo. We could, however, redo sca21 today...

On a happier note, sequencing results came back for sca16,20, 22-25: sca16 and sca20 had mixed inserts inside it, as we expected that there was something wrong those two anyway. Sca22-25, however, came out as perfect reads!!!! Sca15, 17, 18, and 19 didn't turn out so well: the concentration or signals were too low. As of now, Layer 1 has 4 completed, confirmed "good" assemblies, and most likely 7 assemblies that need to be redone.

Layer 1 status

sca15 -- redo 7/22 sca16 -- redo 7/23 sca17 -- redo 7/23 sca18 -- resequencing sca19 -- resequencing sca20 -- redo 7/23 sca21 -- redo 7/22 sca22 -- good/ complete sca23 -- good/ complete sca24 -- good/ complete sca25 -- good/ complete

21 July 2008 (M)
Good: sca16-3, 16-5 sca20-5 sca22-1, 22-3, 22-5 sca23-3 sca24-1, 24-3 sca25-1, 25-5
 * Checked on the colonies from Sunday. Results are as follows:

Bad: sca21 -- all 5 colonies co-transformed.


 * Digestion mapped those that were good. Sca16-3, 16-5, and 20-5 looked a little strange, but the rest of the gel looked absolutely more beautiful than we expected it to be! We sent out the following for sequencing:

sca16-3 (to see what went wrong) sca20-5 (to see what went wrong) sca22-1 sca23-3 sca24-1 sca25-1

We figured that sca16 and sca20 had to be re-assembled, but we first had to amplify our {fimH>} part. We also had to restreak sca21 again to check for co-transformation.

19 July 2008 (Sa)

 * Came in at 2:30pm to assemble the rest of Layer 1 assemblies (7 assemblies total)


 * Digestion mapped 2 colonies of sca18 (4th and 6th) and 3 colonies of sca19 (1st, 5th, and 8th). Sent sca18-4 and sca19-1 for sequencing.

Cici will come in on Sunday to pick 5 colonies each and check for co-transformation.

18 July 2008 (F)
Today:


 * found out that assemblies sca15 had 2 good colonies out of 3; sca17 had 1 good colony out of 3; sca18 and sca19's 3 colonies all co-transformed.


 * miniprepped said assemblies and two tubes of basic parts <phoA! and b1006 each.


 * digestion mapped <phoA! and b1006 basic parts; sca15-1, sca15-3, and sca17-2 were also digestion mapped. We, however, had to redo the digestion, due to the fact that the samples had not digested properly and showed only single-cut bands on our gel; and we used a 4th colony of  again...this time with a positive result!
 * picked colonies for <phoA! and b1006; grew also in media
 * Picked 3 colonies for each of our 4 assemblies, streaking them on their respective double-antibiotic plates and an ACK plate to check for cotransformation.

16 July 2008 (W)
Recap of the past few days and today's work:

Today:


 * Digestion mapped pbad, rbs1-A, and  did not turn out well and had to be re-checked for co-transformation: picked more colonies for that sample; also picked colonies for pbad, rbs1-A, and <HA!.


 * Helped with another set of transfers for purification.

Monday (7/14):


 * Got sequencing results for my batch of 6 prepro basic parts. Five of them came out fine, but the sixth one (K112608) came out as a bad read. Sent colony #2 for sequencing.


 * Helped with purifying another set of transfers.

On another note, we had decided by the end of last week to abandon the "big assembly line" model. Every person was now assigned to make composite parts for one particular portion our project. Cici and I were given the task of making 8 composite parts using the tag basic parts we made some time ago last month. After Jin gave us a run-down of how to approach composite part making on our own, we mapped out our tree, starting out with the basic parts. We determined that we would need 3 layers in our tree to assemble our composite parts. Then, we checked to see which parts had already been transferred to the double-antibiotic vectors that we needed them to be in. Thankfully, all our basic parts had already been transferred into the correct vectors. I picked colonies for some of the said parts from the stock plates and grow them up overnight in media.

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:

First, I fixed up/updated my personal "basic part" documents with the new information from our (Cici's and my) basic parts. Then, I helped organize our list of the needed K numbers, ig numbers, and stock plate numbers for our first set of actual assembly digestions. We ended up setting up for a total of 10 assemblies, once we figured out which parts we didn't have DNA for. An hour after digesting the lefty and right parts with their respective mastermixes (and restriction enzymes), I got to do a purification.

Purification this time was a little bit trickier than before. For one, I was pipetting lefty parts in with the righty parts by hand, and there was definitely a chance that I could mess up. Secondly, our one, precious magnetic plate was being used simultaneously in miniprepping...so, I could not use it! Thankfully, we were able to use a seperate set of "stick-like" magnets, place them between our two rows of tubes, so that the beads in both rows of tubes could move strategically towards the sides (and not sink at the bottom). However, because the stick magnets were taller than the tubes, rendering the tubes incapable of balancing on their own, Bing held the tubes and the magnets while I pipetted. The makeshift "magnet plate" was not completely cooperative, but it served its purpose; and, we managed. We later realized though, that there may have been one righty part that was not placed with a lefty part, but we're not 100% sure... Once purification was done, Jin brought me my new oligo for my basic part that had to be redone! I set up PCR for it, and for 3 other samples as well.

The day ended at 7:15ish with plating for the 10 assembly samples I purified earlier.

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! Another happy discovery I made: the box that Cici and I share for keeping our oligos, parts, etc... was out of room! We are now both entitled to our own boxes - 'twas most exhilarating.

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...). After lunch, we had our pictures taken.

Since Cici left before she could start helping Madhvi with colony PCR later in the day, 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!

Sherine Cheung Back to Berkeley All Notebooks