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Template:Team:UC Berkeley/Notebook/Molly notes
From 2008.igem.org
Molly's Notes
07/16/08
Nevermind about the tree below. I have a new one that I have in a spreadsheet on my labtop that I'm using.
Today, I picked from stock plates/grew up ig9, ig28, ig26 and ig122 in culture tubes. They are upstairs in the warm room shaker. Tomorrow, I will mini-prep them and digestion map them. I can also start assembly with ig28 and ig26.
I also am doing transfers of molly2, molly4-11. However, molly10 (rbs.phoApp>) was put into K/C, and I need to put it into C/K, so I'm using Sherine's PCR digested product and doing a ligation using 6.5 ul water, 1 ul ligation buffer, 0.5 T4 DNA ligase, 1 ul [C/K] digested with EcoRI and BamHI and 1 ul of PCR digested product.
07/15/08
Didn't come into lab today.
07/14/08
So I didn't know that the shaker in our lab would dry out the petri plates, so the Cam, Kan and Amp plates were all dried out. The Spec plate showed background/contamination, although the transformed cells did grow better than those that were not transformed.
The petri strips plated yesterday seem to have grown fairly well. I prepared four petri dishes to pick two colonies each. One is a PR#18: A&B, 1&2 which is a C/A plate. Rows A1 and B1 represent the first colonies picked from the positions as specified by the PR#18 spreadsheet. Similarly, there are plates PR#19: A&B, 1&2 which is a A/K plate, and PR#20: A&B, 1&2 which is a K/C plate. The fourth plate is a Spec plate to check for co-transformation. It is labeled PR#18: A&B, C&D (representing that rows A&B are for colony 1, and C&D are for colony 2), PR#19: E&F, G&H and PR#20: I&J, K&L. They are similarly distributed as they were on their respective PR plates. There are also 3 96-well blocks of C/A, A/K, and C/K TB, and all are analogous to the PR#18-PR#20 plates. Aron and Sherine picked, streaked, streaked, and plated. All are upstairs in the warm room.
I also transformed an assembly test from Bing into Mach1 cells and plated them on a Spec plate with a smiley face on it which is upstairs in the warm room as well.
We decided that I would be in charge of the Gateway vector. Here is the assembly tree Clothos spit out:
a.b.c.d.e.f.g: { a.b.c.d.e.f.g[C/K] a.b.c[C/A] a[C/K] b.c[K/A] b[K/C] c[C/A] d.e.f.g[A/K] d.e[A/C] d[A/K] e[K/C] f.g[C/K] f[C/A] g[A/K] } a.h.i.j.k.l.m.n.o.f.g: { a.h.i.j.k.l.m.n.o.f.g[A/K] a.h.i[A/C] a[A/K] h.i[K/C] h[K/A] i[A/C] j.k.l.m.n.o.f.g[C/K] j.k.l.m[C/A] j.k[C/K] j[C/A] k[A/K] l.m[K/A] l[K/C] m[C/A] n.o.f.g[A/K] n.o[A/C] n[A/K] o[K/C] f.g[C/K] f[C/A] g[A/K] }
I want to re-use these parts:
ig141 (K112214) [C/A] – in 7/3 Stock Plate #2, G7 (hasn’t been colony PCRed or digestion mapped) ig1085 (K112230) [C/A] – in 7/13 Stock Plate #1, B1-12 (in process of growing up) ig18 (K112227) [K/A] – in 7/3 Stock Plate #4, D8 (no colonies)
I won't be here tomorrow, so I picked a colony from 7/3 Stock Plate #2, G7 (ig141) and grew it up in about 5 ml of 2YT C/A. It is in a glass culture tube and is in the shaker upstairs. (It was put in at about 7:30 pm) Tomorrow, Jin will mini-prep my sample and do digestion mapping to make sure it is correct. Also, Madhvi is going to colony PCR it. Tomorrow we also need to screen 7/13 Stock Plate #1, B1-12, and if it is good, similarly grow it up.
07/13/08
Came into lab today. Apparently the mini-preps Jin did yesterday didn't work out, so we're re-growing the colonies again in richer broth and will mini-prep them again today. I also tested the competent Mach1 and DH10B cells that were prepared yesterday. They are growing on Cam, Kan, Amp, and Spec plates. Some were transformed with 1 ul pBjh1600 plasmid, rescued for about an hour and plated on the Spec plate. They are in the shaker in our lab.
Jin needed some samples mini-prepped today, so I did 10/14 since 4 were redder than they should have been. However, I ended up forgetting to do the extra 1 min spin to get rid of all the ethanol before I eluted with water, so I put them in the 50 deg heat block to hopefully evaporate some of the ethanol. (Sorry, Jin!)
Bing is also doing 56 more transfers today.
07/12/08
Kayaking trip! :)
07/11/08
Today we were filmed! Ah! We also did our first round of assembly on the 10 pairs that actually worked. I transformed them into Mach1 cells and rescued them, but then had to leave so someone else plated them for me. See google doc Post Rescue Plates, sheet 7/11 for what was plated.
07/10/08
I came in an everyone was mini-prepping, so I joined the assembly line. We diluted with 25 ul of water, which Chris says is too little (the minimum is 30 ul). So... he's trying out the magnetic bead mini-prep method with 8 extraneous samples to see if it would work. We also had a mini-meeting today and I showed my animation. Almost done! Jin got us pizza (thanks Jin!) and afterwards I picked colonies from stock plates 1-4. They are in three 96-well blocks which are on the shaker upstairs in the warm room to grow overnight.
07/09/08
Bing took the samples I grew in culture tubes and put them in the fridge. I spent the majority of the day deciding which enzymes to digest each sample with and updating the lengths. We also had a modeling meeting.
07/08/08
Picture day! Today I helped out with whatever needed to be done. Most notably, I picked and grew up colonies that had been screened on Spec and their respective antibiotic plates. They are in the warm room shaker to grow overnight.
07/07/08
Today involved a lot of updating and working with spreadsheets. However, I did transform 21 samples (See Transformation Plate #6) and plated them on petri dishes instead of strips since we ran out. They are upstairs in the warm room.
07/06/08
It turns out that we only have to repeat about 30! All the rest had colonies that grew. Jin is testing today for false positives (i.e. has both the parent and assembly vectors). I transformed and plated these 30 samples (except for the sample in D1 Aron said was contaminated). See the Google docs. Bing is making more 2YT for me today (thanks!)
07/03/08
Busy busy busy... Yesterday's 48 samples didn't work, so we need to do those over again. I also transformed/plated two other 96-well plates worth of samples (see the Google Docs for which ones). We need more petri strips.
07/02/08
Today I helped design oligo's for {<phoA>} (part K112233). See my construction file.
I also transformed the first 48 (or maybe less seeing as how some were empty) samples. I transformed and plated them all. See the google doc's for what sample is in each well. These were all either CA or AK. They are up in the warm room along with the samples I plated for Bing tonight.
06/30/08
Today I worked a lot on the assembly protocols. I also created a bunch of spreadsheets for each person to use to keep track of where each of their samples are. I'm learning flash... slowly. It seems to be a much better option for the animation though. I also designed construction files for {<barstar!} and {rbs.barstar!} (parts K112231 and K112232). I'm not sure if/when Jin is planning on ordering the oligos, but he said they looked pretty good.
06/26/08
I took the stock plates out of the warm room and put them in the fridge. I also got the rest of my sequencing data. Clone 2 for both K112201 and K112204 were both perfect, so we're going to use those.
06/25/08
After our mini-meeting, I got my culture tubes growing two colonies each of K112201 and K112204 and mini-prepped them a long with some of Bing, Aron, and Madhvi's samples. I should be getting sequencing data tomorrow. K112201 clone 2 will be MEA033, K112201 clone 3 will be MEA034, K112204 clone 2 will be MEA035, and K112204 clone 3 will be MEA036.
I also worked a lot today on the "blown away" logo idea. Terry suggested making an animation, so I will also work on that.
Remember to take the stock plates out of the warm room!
06/24/08
I got sequencing data! K112200 (MEA026), K112205 (MEA029), K112207 (MEA030), K112210 (MEA031), and K112212 (MEA032) are all perfect. However, K112201 (MEA027) has a point mutation in it changing GCG (Ala) to GTG (Val). K112204 (ME028) had a mutation in its a~ site from TGCG to TGTG, but more importantly had a deletion of a G later on in the sequence.
I picked two colonies each from K112201 (clones 2 and 3) and from K112204 (clones 2 and 3). I also made a stock plate which is parafilmed and in the fridge. The colonies are being grown in 4 mL of 2XYT with spec and are in chris's shaker upstairs. Tomorrow I need to mini-prep them and send them in for sequencing.
The colonies from the BX_Cell Stock Plate #1 helped Bing grow up yesterday were mini-prepped today. I organized a series of final 96-well stock plates #3.1 and #3.2 (see google docs) and designated wells for everyone's basic parts. I added all the mini-prepped samples to their appropriate wells, and added all my other correct plasmid stocks to the appropriate wells. The only two I am missing are K112201 and K112204, which are being grown up tonight and will be ready for sequencing by tomorrow.
06/23/08
So things are switched around for reads of K112207-212. The respective read files are: AL305, MA212, MA209, MA211, MA210, and MA208. MA209 (K112209), MA210 (K112211), and MA212 (K112208) are perfect reads, while AL305 (K112207) , MA208 (K112212), and MA211 (K112210) were contaminated with a second template by the sequencing company. So I told Jin that these need to be re-sequenced and I will collect the plasmid samples from Stock Plate #1 and send them in to be sequenced along with K112200, K112201, K112204, and K112205 that Bing mini-prepped for me yesterday.
I made some changes on the iGEM DNA Stock Database google doc to account for the sample switch-around. I switched K112217 with K112218. Also, all the pelB parts were switch around, so K112228 is actually K112229, K112229 is actually K112230, and K112230 is actually K112228. These were changed on google doc appropriately. I also switched these around on BX_Cell Stock Plate #1.
I also helped Bing grow up a bunch of the clones from BX_Cell Stock Plate #1 that were initially contaminated with RNA so that we would have a plasmid stock for composite part making later on. They are in two 24-well block plates incubating.
06/22/08
Bing mini-prepped K112200, K112201, K112204, and K112205 for me. (Thanks!) I also got my reverse sequencing data that they re-ran with G00101 for K112207-212, but they're kinda funky. I'll ask Jin tomorrow if things are switched around.
06/21/08
Madhvi picked colinies for K112200, K112201, K112204, and K112205 for me. (Thanks!)
06/20/08
I retrieved my gel-isolated PCR products of K112200, K112201, K112204, and K112205 (using the real mea002 oligo!) and digested them, did a zymo clean-up, ligated them with twice the recommended amount of pBca1256 re-digest since it was dilute, transformed and plated them. They are incubating in Chris's lab.
I also got sequencing data on K112207-12,229. K112229 turned out to indeed be the sequencing for K112230, and it was perfect aside from a strange deletion of A in the EcoRI site, but it looks like it's just a bad read error. K112207-12 were supposed to be run with G00101, but were instead run again with ca998... but they do have enough plasmid to do another reverse read, so that's good. I'll be expecting that data soon.
Today, I also did a mini-prep on K112219, K112220, K112223, and K112224 colonies I picked/grew up in a culture tube yesterday. The samples will be sequenced under the names MEA219-224.
06/19/08
I checked plates K112205, K112219, K112220, K112223, and K112224 today and threw out K112205 since it used the faulty mea002 oligo. LOTS (around 50% in some cases) of the colonies are red (parent vector), indicating Aron's vector digest probably wasn't all that pure. The K112212 plate has a ton of very very tiny colonies growing, so we parafilmed it an put it in the fridge. The glass culture tube was dumped into the 24-well block with Bing and Aron's samples and Bing mini-prepped them. At the end of the day, I picked colonies from K112219, K112220, K112223, and K112224 and grew them in 5 mL of 2XYT/Spec in glass culture tubes and left them overnight in Chris's shaker.
Because there were many red colonies, we decided to re-digest the vector digest that Aron made with the newer digest that Bing and Aron made. We let them digest for 2 hours instead of 1. Then I ran all of the sample on a clone-well E-gel to isolate the digested vector. The bands were surprisingly light and comet-like, suggestive of over-loading. However, Chris looked at it and said it looked fine, so I went ahead and isolated that and put it in the freezer.
Also, today Chris gave us a tutorial on how to use Adobe Illustrator and someone took a group pic.
My new oligo mea002 came today! I diluted it to 100 uM and made a 10 uM stock with 5 ul of oligo and 45 ul of water. I used this to set up a PCR of K112200, K112201, K112204, and K112205 and ran them with two of Aron's samples with program 55. Aron stayed late today and said he would gel-isolate my product as well as his own and store mine in the freezer. (Thanks!)
06/18/08
So I took out my PCR products, ran/isolated them on an E-gel. I took my K112205 sample out of the freezer and included them with my isolated K112219, K112220, K112223, and K112224 samples in digestion, zymo clean-up, ligation, transformation, and plating. Chris had run out of his digested pBca1256 vectors, so we used the ones Aron digested the other day. We included one of Bing's samples that had previously worked as a control. These were left to incubate at 37 C in Chris's lab.
Terry came in today and talked about the modeling of holin and antiholin. I still need to start looking for papers and reading over the ones that Terry already found.
Sequencing data came in today as well. All the integrase reverse-sequencing reads (done after the sequencing lab re-amplified the product we already had) gave bad data 200 bp after the oligo, but were otherwise perfect. We mini-prepped the stock clones (K112207-K112211) but there wasn't any for K112212. So we took the plasmids from the clone we sent in originally and re-transformed them back into bacteria. Part of the transformation was plated, and part was grown up in a glass culture tube containing spec. Both are in Chris's lab. We also picked a new (third) colony for K112229 since the sequencing data was bad again for this. K112229 is in A1 of a 24-well block along with Bing and Aron's stuff. K112207 is in B3, K112208 is in B4, K112210 is in B5, K112209 is in B6, and K112211 is in C1.
The sequencing data for K112200, K112201, K112204, K112216, and K112229 also arrived today (MA001, MA002, MA003, MA005, and MA004 respectively). The read for colony 2 of K112204 was a bad read, as was the read for K112229. K112216 proved perfect. K112200 and K112201 showed a common point mutation changing Asn to Lys, which was also identical to the mutation in the first clone of K112204. So I decided to check out their common oligo, and it turns out that Jin had erroneously added an extra C to the 3' end of this reverse oligo which cased the fatal point mutation from a T to a G. <shakes fist indignantly> This means I will have to start over on K112200, K112201, K112204, and K112205.
06/17/08
My new (correct) oligo mea014 arrived today, so I set up PCR for K112219, K112220, K112223, and K112224. Most of the day otherwise was spent discussing composite parts and such. Bing and Aron picked new colonies for the ones that didn't work (for me that was K112200, K112201, K112204, K112216, and K112229).
06/16/08
Today I analyzed all of the sequencing data that Bing and Jin sent in over the weekend. See my sequencing log page for the results. Some of the sequencing files seemed to be switched around, so Jin is having them re-sequence some of them. The ones that were "perfect" were transformed into competent cells. All of the integrase basic parts were also sent back to be sequenced with the reverse oligo G00101 since they're over 1000 bp. The integrase template also seemed to have a silent mutation, since the exact same one showed up in all 6 basic parts.
Bing and Aron also picked other colonies for the sequencing data that didn't work.
06/13/08
So I ran K112205, K112219, K112220, K112223, and K112224 with and without DMSO on E gels. K112205 with and without DMSA both worked, and are in the freezer in tubes labeled A6 with and without a black filled-in dot for with and without DMSO. However, none of the others did work. We checked it out and discovered that mea014 was not copied and pasted correctly in the oligos to order spreadsheet, so it was actually just another forward oligo rather than the reverse that was crucial to all four parts that didn't work. Jin will order it on Monday in case anyone else needs more between now and then. I've decided to wait on my successful PCR with K112205 until I have the other four done.
All of my plates looked good (even the contaminated one, K1122209!) so we made 2YT media, picked colonies, and put them in a 96 well plate-thing with 1 mL of media each. We put it on the shaker table in the 37 C room to grow overnight. Tomorrow, Bing will come in and do a miniprep/send them off to be sequenced.
06/12/08
Today was a busy day...
First, I ran all 24 of my PCRed parts (K112200-K112224) again and got acceptable products for all but K112205, K112219, K112220, K112223, and K112224. So, I ran these five again with and without DMSO with program 45 and will run an E gel tomorrow. The rest of my samples were digested with EcoRI and BamHI, cleaned up with the zymo kit, ligated with pBca1256, and transformed into competent cells. It should be noted that K112209 was submerged in the ice-water bath before heat-shock, and was thus probably contaminated. My wobble parts (K112225-K112230) were similarly prepared and plated. Finally, all were plated using sterile glass beads onto plates with Spec and placed in the 37 deg room to grow overnight.
06/11/08
All of my oligos came today (yay!) I successfully made all my wobble prepro parts (i.e. K112225-K112230) today, purified them on an cloning E-gel (next time remember to keep filling the second wells to get the max amount of product!) I got about 15 uL of each product. Next, these were digested with EcoR1 and BamH1, and incubated at 37 deg C for 1 hour. Finally, the DNA was purified using the Zymo kit.
The master mix used for all 24 of my other parts (K112200-K112224) had too much of the dNTP mix, and running it on an analytical E-gel showed that the was no PCR product. At the end of the day, we tried again and put them in the thermocyclers to be purified tomorrow.
