Template:Team:UC Berkeley/Notebook/Molly notes

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Molly's Notebook


Molly's Notes



(Moved to physical notebook)


I digested the four minipreps of pK112246 with BglI/XbaI. I am expecting band lengths of 1171/5597. It looks as though #1, 2 and 4 worked. I will send out #1 for sequencing of the att sights.

I also had Jin transform pK112128 #2, pK112245 (the one that was sequenced, #2) and pK112246 #1 into TG1 cells to rescue and plate each on CA.


The sequencing data came back for pK112245 and it looks like the attR1 and attR2 sites are both perfect. Bing mini-prepped my four cultures of pK112246 and I will BglI/XbaI digest them tomorrow. (Thanks Bing!)


I miniprepped the five colonies from pK112245 and the one from pK112246 and digested them with BglI/XbaI. If there is no part in it at all, the bands should be 1171/2804. pK112245 is 1171/4985 and pK112246 is 1171/5597. Only colonies 2,4 and 5 from pK112245 looked like they assembled, while pK112246 looked questionable. I sent in colony 2 for sequencing the attR1 and attR2 sites. I will pick the four colonies that grew on my plate from yesterday and miniprep/characterize them tomorrow.


I came in today and there was only one colony growing for pK112246, and around 10 for pK112245 (of which I picked 5). These are growing up in CA LB in the 30 deg room in TG1 cells. In case none of these are correct, I used the last of the digested materials to try and re-ligate these again. They are on a CA plate in the 30 deg room.


So I've been super busy, but for the first time I got promising results for gateway. So far, I have the two assembly vectors with {prom.xis.int!} and {prom.xis.int.ihfA.ihfB} which are pK112245 and pK112246 respectively, and the two entry vectors again with {prom.xis.int!} and {prom.xis.int.ihfA.ihfB} which are pK112247 and pK112248 respectively. I also have the entry vector for scheme 1 with a fixed attR2 site in it thanks to Christie; it's called pK112128#2. I got sequencing data back this morning telling me that there was still the attR2 mutation in pK112245 and pK112246, so I'm going to take pK112128#2, and PCR it with mea065 and mea066 so I can digest and ligate my {prom.xis.int!} and {prom.xis.int.ihfA.ihfB} parts in. Also, I did an experiment in which I transformed in all the entry vectors directly into MC1061 and plated on CA to make sure they don't have CA resistance. Nothing grew for pBca1256, pK112247, or pK112248 so it looks like its okay! However, I also did this for the assembly vectors pK112128#2, pK112245#4, and pK112246#5, and it seems as though the ccdB in pK112246#5 is really bad since I got a lot of colonies... The other two only yielded about 6 colonies each, meaning the ccdB in these is (for the most part) working in preventing most background from showing up.


Yay! The sequencing showed that the temperature sensitive promoter assembled with K112237 to make K112244! I'll have to wait until late-ish tonight to pick colonies, assuming there will be some to to pick! <crosses fingers>


I ran the two PCRs from yesterday (mea062+mea061 on mv36, and mea062+mea060 on K112244) out on a gel, and the bands looked good. I gel purified these and zymoed these plus the two that worked from yesterday. Then, I digested pBca1256 and pBjh1601CA each with SpeI/MfeI, and mv36 and K112244 each with EcoRI/NheI. After zymoing and eluting with 6.5ul water, I set up ligations between pBca1256+mv36, pBca1256+K112244, pBjh1601CA+mv36, pBjh1601+K112244. Next, I transformed them into TG1 cells, rescued in no-antibiotic LB in the 30 deg room for 1 hour, plated the two with pBca1256 on Spec and the two with pBjh1601CA on CA plates which I put in the 30 deg room to let grow.

I also sent out P_ts + K112237 (=K112244) for sequencing with ca998 to make sure the promoter attached in front.


So over the weekend, Christie did four PCRs for me: mea063+mea064 on pBca1256, mea065+mea066 on pBjh1601CA, mea062+mea061 on mv36, and mea062+mea060 on K112244. However, only the first two appeared to have worked (which I melted in ADB buffer and put in my box), so I set up the second two PCRs over again.


I ordered oligos mea060-mea066 (NheI-reverse ihfB!, NheI-revers int!, EcoRI-forward pAH57 promoter, SpeI-forward attL1, MfeI-reverse attL1, SpeI-forward attR2, MfeI-reverse attR2, respectively).


I went ahead and mini-prepped P_ts + K112237 (=K112244). It's in my box, and it's from righty.


I picked 8 colonies from the {pAH57 promoter}{rbs.xis.int!}{rbs.ihfA!}{rbs.ihfB!} (K112126 + K112237 = K112244) and ran colony PCR on all of them as well as on K112237 alone for control purposes. Only #3 seemed to have assembled, so I grew it up.


Today I digested K112237 with BglII/XhoI (L,3415+995) and K112126 (the temp sensitive promoter) with BamHI/XhoI (L,1878+1486) to try and assemble them. I got the correct bands, but it was hard to separate 1878 from 1486, but I tried anyway. I zymoed and eluted with 9ul water, ligated with 7.5ul DNA/water, 1ul NEB2, 0.5ul BamHI, 0.5ul BglII, and 0.5ul DNA ligase. Finally, I transformed them into leftover righty cells and plated them on a Spec plate which is growing in the 30 deg room.

I also ran colony PCR on mv37 and mv39. mv 37 stuff is in the 30 deg room and the mv39 stuff is in the shaker.


Yay! Colonies to pick! Also, the plate from 9/11 also had some colonies but they're probably just contamination... I picked 8 from the 9/12 plate, and 4 from the 9/11 plate and ran a colony PCR with ca998 and the reverse oligo for <b1006>. Hopefully I'll get a bright band around 2100.

I also took out pG80K0-mv23 EcoRI/BamHI, pBca1256-Bjh1233 EcoRI/BamHI, pBca1256-Bjh1245 BamHI/XhoI, and pBca1256-attR2 BglII/XhoI digests out of the thermocycler for Madhvi and ran them out on a gel. I was able to get all the bands except that for mv23; only the 2314bp band was visible, so I just cut out the area where the 506bp band might have been... I digested again with the same results, so I went ahead and just zymoed and ligated (with background subtraction for the Bjh1245 + attR2 assembly). mv23 + Bjh1233 were transformed into pir+ cells, and Bjh1245 + attR2 were transformed into Lefty.


There are no visible colonies on my plate from yesterday, so I mini-prepped <b1006> pBca1256 that I grew up yesterday in Righty, digested it with BglII/XhoI along with K112237 digested with BamHI/XhoI, gel purified, ligated with background subtraction and Christie rescued/plated it for me (thanks!) Hopefully I'll have colonies tomorrow...


I mini-prepped the 8 colonies from K112237 + ccdB = K112241 from yesterday along with {rbs.xis.int!} in pBca1256 in Lefty and in Righty that Madhvi transformed last night. I digested colonies 1-8, K112237, and two samples of {rbs.xis.int!} in pBca1256 in Lefty. I will expect the colonies to be at 3772 + 1486, and K112237 to be at 3003 + 1486. Hopefully I will finally get my *2287* + 1486 bands from K112234 so I can continue with assembly. None of this worked... bands of 8 colonies were weirdly bright, but at the same lengths as K112237, and the bands from K112234 were too light to use... Jin says to abandon this scheme.

I went ahead and took out <b1006> from DNA Stock Plate #3.1, H9 and amplified it in righty. I think the general plan now is to make the whole {pAH57 promoter}{rbs.xis.int!}{rbs.ihfA!}{rbs.ihfB!}{b1006} as well as {pAH57 promoter}{rbs.xis.int!}{b1006} and then stick it either behind or in front of a plasmid that already has ccdB in it. Madhvi has {pAH57 promoter}{rbs.xis.int!}, and is going to attach the terminator onto the end of it, but I'm going to try and make {rbs.xis.int!}{rbs.ihfA!}{rbs.ihfB!}{b1006} and then stick on the {pAH57 promoter}. I digested some <b1006> from the stock plate and tried to do a BglII/XhoI + K112237 BamHI/XhoI assembly with background subtraction, but the bands were really light, so I wouldn't be surprised if it didn't work. I'll probably have to just use the amplified <b1006> what I will mini-prep tomorrow.


I picked 8 colonies from the K112237 + ccdB plate and ran colony PCR with the forward oligo for {rbs.ihfA!} and G00101. I expect to see bands at around 1030 bp if the assembly worked, and at only about 630 bp or 400 bp if it didn't work. There was probably something wrong with the oligos because there were no bands... I'll grow them all up anyway and mini-prep/digestion map them tomorrow to be sure.

I also mini-prepped molly 19-1 (K112237) and ccdB (molly4) that I reamplified yesterday. I will digest molly4 and molly14-3 in BglII/XhoI, and two samples of K112234 in BamHI/XhoI. molly4 should be *1878* + 995, molly14-3 should be *2003* + 995, and K112234 should be *2287* + 1486. I also did a BamHI/XhoI digest on the molly19-1 I reamplified just to make sure it was correct, and this should be 3003 + 1486. molly19-1 looked correct, and so did the bands for the righty parts, but here was apparently no DNA in the K112234 MP I used, so I'm re-digesting them now.


I digested K112237 (pBca1256) and ccdB (pBca1256) which I got from Dan with lefty and righty enzymes, respectively. Molly19-1 gave blurry but light bands, but I think they were okay. I gel purified, zymoed them together and ligated with BglII/BamHI/Ligase/NEB+ATP. They were transformed into TG1 cells, rescued, and plated on a Spec plate. I also simultaneously amplified molly19-1 and ccdB in TG1 cells, which are in culture tubes in the shaker.

Tomorrow, I hopefully will have colonies which will need to be picked and characterized. I also will start making {rbs.xis.int!}{b1006}{ccdB} and {rbs.xis.int!}{ccdB} tomorrow.


So Jin wanted me to try digesting again to see if all the bands show up clearly, so I digested molly19-1, K112238 #1, #12 and #13 with EcoRI/XhoI and ran them out on a gel (see "molly digest 9-8-08" Ladder, molly19-1, 238 #1, #12, #13). I expected molly19-1 to have bands of 3503 and 986 bp, and K112238 to be 4239 and 986 bp. They're really weird bands... I don't think it worked... Boo...

I also cut out bands for and did background-screening assemblies to make Bca1109 + rbs.xis.int! and K112126 + rbs.xis.int!. I transformed ligated them and transformed into Lefty cells which I accidentally started rescuing at 37 deg C, and after about 10 min moved K112126 + rbs.xis.int! into the 30 deg room. I plated them both on a Spec plate and they are both in the 30 deg room to grow up overnight.


I came in today and mini-prepped TrfA Strain w/Forigin #1 and #2, mv21 in CA, mv20 in KA and rbs.xis.int! in 1256 and eluted with 40ul water each. I digested 4 aliquots of rbs.xis.int! with BglII/XhoI, mv20 and mv21 with EcoRI/BamHI, and finally K112126, Bca1109, Bjh1377 and Bjh1380 with BamHI/XhoI. I rand them all out on a gel "molly mv assemblies 9-7-08" which are in the following lane order: mv20, mv21, ladder, rbs.xis.int! #1,#2,#3,#4, ladder, Bjh1377, Bjh1380, K112126, Bca1109, and ladder. mv20, K112126, and Bca1109 seem to not have worked, although there is DNA there. I digested them again and ran them out on "molly mv assemblies redo 9-7-08" which is mv20, ladder, K112126 and Bca1109. mv20 doesn't have any DNA which is weird... and the other two look like single cuts. We need to figure out what's going on with these...

Anyway, I did cut out the bands for mv21, rbs.xis.int! #1-4, Bjh1377 and Bjh1380. I combined Bjh1377 and Bjh1380 with the bands cut out from rbs.xis.int! #1-2. (rbs.xis.int! #3-4 are in two separate tubes in anticipation of zymoing them with K112126 and Bca1109.) I zymoed these 3 and eluted with 6.5ul water each. I then added 1ul of pBca1256 EcoRI/BamHI digest to 1ul mv21 zymo and ligated. Bjh1377 + rbs.xis.int! and Bjh1380 + rbs.xis.int! were both ligated with 1 ul NEB+ATP, 0.5ul of each BamHI, BglII, and T4 DNA ligase to do background screening. All three were then transformed into Righty cells.


Richard said neither ca998 nor molly022 worked. He will re-sequence if he has more template.


Only the reverse read worked, and it looks like there's no {b1006}{ccdB} attached to the end, but I want to know why it's a little bit bigger than molly19#1. I sent out two more aliquots of K112238#1 along with molly022 (forward oligo for rbs.ihfA!) to be sequenced with molly022 and ca998.


I digested 3ul of molly19#1 with DraI/XbaI and ran it out on a gel. I expect band lengths of 4108 and 381... which I didn't get. I only saw one band at about 2500 which is very strange, so I'm going to do an EcoRI/XhoI digest on all 4 samples and run them all out again so I can compare them side-by-side. See "IMG_0764" in which first is ladder, then molly19#1, then K112238#1, K112238#12 and finally K112238#13. The latter 3 are clearly larger than molly19#1, so it may have worked! I sent in K112238#1 for sequencing with G00101 and mea013. <crosses fingers!>


Today I mini-prepped the 3 colonies I grew up of K112238 last night and digested them with DraI/XbaI so that if they did infact assemble, I should get bands at 4108 and 1117, whereas if it didn't work, I should get bands at 4108 and 381. I also digested molly19#1 with DraI/XbaI, but Dirk forgot to run it out as well so I'll do this tomorrow for comparison's sake.


Nice colonies on K112238 plate! I picked 16 colonies and ran colony PCR with the forward oligo for {rbs.ihfB!} and the reverse oligo for {<b1006>}. I should get a band around 380 bp if it assembled correctly, and no bands if not. The bands were really blurry and so it doesn't look like any of them assembled... boo. But Jin said to pick 3 of them, grow them up anyway and digestion map them just to make sure. I chose colonies #1, #12 and #13.


I digested my zymoed dbbs of molly19#1 with BsaI, DpnI and BamHI, zymoed, and ligated with molly14-3 dbbs. I then transformed them into TG1 cells, rescued them and plated them on a spec plate. It's in the incubator.



I decided to try dbbs over again on molly19#1 transfer into 1256. I ran 4k55 with ca998 and G00101, and then ran it out on a gel. The band should be about 2176 bp. It was, so I cut it out, zymoed and eluted with 90ul of water. It's in my box.


All the colonies picked from mv13 are not E. coli, nor is mv5 colony #2. The rest were mini-prepped and I digested mv5, mv21, OriV, and promoter with EcoRI/XhoI and ran them out in a gel (see "molly eco-xho digest 08-28-08" in which 3 colonies each were run out in this order, with the ladder in the middle). The bands looked weird... I couldn't tell if the promoters were correct, so I assumed they were and digested #1. I also ran a gel with mv27 #1-3 alongside R6K digested with EcoRI/XbaI (see "molly eco-xba digest 08-28-08", ladder, #1-3, R6K) but the bands were really weird and the gel warped the ladder... mv27 #2 looked the closest to being correct, but it looked like none of them really worked... SO I went ahead and set up the Lefty/Righty digestions with ig1082 KC/ig161, 1256-KA #1/mv27 #2, Bjh1245-1/promoter#1, and R6K/attR2 rev.

Jin wants to re-PCR OriV, remembering to used DpnI.


I picked 3 colonies each for mv5, mv13, mv21, mv27 (all transfers into pBca1256), OriV entry vector, and temp. sensitive origin from pAH57. They are growing in LB Spec and there is a stock plate in the incubator (although I forgot to streak OriV entry vector colony #2).


I miniprepped TG1 OriV in pBca1256 w/PUC #1 and #2 and digested 3ul of each with BamHI and XhoI. The bands should be 1896 and 2040, but if they are 1142 + 2040 it's only the OriV entry vector, and if they are 1486 + 1896 it's just pBca1256. They both look fine. I ran a 4K55 PCR with each.

I also digested mv5, mv13 and mv21 with EcoRI/BamHI, ran them out on a gel, zymoed, ligated with pBca1256 and transformed into lefty.


Madhvi and I just talked about what needs to be done. We will start tomorrow.


Only about 7 colonies grew, and I didn't have any of the oligos for Bca9471 to do colony PCR with, so I went ahead and picked them and hope digestion map the mini-preps tomorrow.


Bah, the plate with Bca9471 + {rbs.xis.int!}{attR2} is contaminated... I'll start assembly over again... This time I only digested 3ul Bca9471 because there were a lot of single cuts. All the rest is the same as yesterday. Jin plated it for me (thanks!) so it's on a Spec plate in the incubator.


I mini-prepped the {rbs.xis.int!}{attR2} sample that worked. I digested it with BglII/XhoI (*2920*, 955) and I digested Bca9471 with BamHI/XhoI (*5313*, 1486), gel purified them, and zymoed them together, eluting with 9ul water to get about 7.5ul DNA+water. I then added 1 ul NEB2+ATP, 0.5 ul BglII, 0.5 ul BamHI and 0.5 ul ligase. They are transformed into Lefty cells and are growing on a Spec plate.

I also picked 10 colonies from the Spec plate I grew up with the dbbs parts to make K112238. I am running a colony PCR with the forward oligo for {rbs.ihfB!}, mea013, and the reverse oligo for {b1006}, dv022. If I get bright bands at around 360bp, the ligation was successful. The bands were too blurry (see "molly colony PCR on K112238 8-21") meaning it probably didn't work since it is likely just the oligos in the band... Jin is going to grow up colony #1 for both molly19#1 and molly14#3 in 1256 and try to assemble it.


I came in today and digested the {b1006}{ccdB} dbbs that I zymoed yesterday with BglII, XhoI and DpnI. I then ligated it with the 19-1 in 1256 dbbs from a while ago and transformed into TG1 and plated on a Spec plate. Tomorrow I need to pick colonies (and do colony PCR to ensure junction is made?). Hopefully I will get colonies this time that when digested with BglII/XhoI will give bands of 4230 (not 2778 or 2003) and 995.

I also picked 8 colonies from the {rbs.xis.int!}{attR2} and ran colony PCR on it with ca998 and mv39 (the reverse oligo of attR2). Afterwards, I will run it out on a gel and hopefully get a band of 1500bp for at least one of the colonies. The third colony had an extremely bright, clear band at 1500, while colonies 1 and 7 also had light bands at 1500. Most contained a band at around 300 (attR2 only). I am growing up colony #3 in a culture tube in the shaker and will mini-prep it tomorrow.

Today, I also tested whether or not BamHI and BglII enzymes worked in NEB2 Buffer + cell lysate. I tool a tube of Righty 1311 and a tube of Lefty 1311 and diluted it 100x with LB Amp (50ul cells to 5 mL LB Amp) and let them grow up in the shaker for 2-3 hours until they reached midlog. Next, I took 1 ml of each Lefty and Righty, spun it down, added 10 ul NEB2 (10X) and enough water to bring the total volume up to 90ul. Then I vortexed to mix, and added 10ul 20% arabinose, making the total volume 100ul. After leaving them in the shaker for 1 hour, not very viscous. I split each into two tubes, giving you a total of 4 tubes, 2 lefty and 2 righty. To the first lefty and the first righty, add 0.5ul BamHI. To the second lefty and the second righty, add 0.5ul BglII. Then lightly vortex just to mix and again but they were still not viscous! Incubate at 37 deg C for 1 hour. Not viscous. Finally, run 20ul of each sample on a gel and take a picture.


I came in and mini-prepped/digestion mapped (BglII/XhoI) (rbs.xis.int! in 1256). The bands look fine, so I digested with BamHI/XhoI to gel purify the 2287bp band. After zymo-ing it, I ligated with attR2 and transformed into righty cells. They are on a Spec plate on the 4th floor. Tomorrow, I will pick colonies and do colony PCR with the reverse oligo for attR2 to make sure they joined together.

I also am running dbbs over again on {b1006}{ccdB} (aka K112236 or molly14 #3). I used ca1132R and ca998 and ran it on 3K55. After, I will run out some of the PCR on a gel to make sure there is a 2088 bp band. I cut out the band, and gel purified it with zymo, eluting with 90 ul water. Tomorrow I need to digest this and again zymo, then try ligating with molly19-1 in 1256 dbbs again.


Turns out Madhvi picked the wrong colony #3... She took it from the 8/2 Mach 1 plate of {rbs.xis.int!} in 1256 which were contaminated. I went ahead and picked the correct one today, and it is growing up in Chris's shaker.


Yay! Colonies grew! Hopefully I've finally assembled K112238! I picked 12 colonies which I streaked on a Spec plate, grew in culture tubes, and ran colony PCR.

Chris also wants me to start making {rbs.xis.int!}{attR2} as well as {rbs.xis.int!}{rbs.ihfA!}{rbs.ihfB!}{attR2}. I took molly19-1 transfer into 1256 and rbs.xis.int! in 1256 and digested both with BamHI/XhoI, and took attR2 and digested with BglII/XhoI. However, I did not see any DNA in the first lane of "molly " which was {rbs.xis.int!}... The other three were gel purified and {rbs.xis.int!}{rbs.ihfA!}{rbs.ihfB!} and {attR2} need to be ligated tomorrow.

Madhvi digested {rbs.xis.int!} again, but still no DNA. No transformations worked. Also, the dbbs didn't work... see image "molly dbbs for k112238 8-14," all the bands are around 700 bp, which is what {rbs.xis.int!}{rbs.ihfA!}{rbs.ihfB!} alone would give... Damn. Need to try again.


The two colonies I picked for both molly19 #1 and molly14 #3 both were fine (i.e. they grew on Spec, not on ACK), so I mini-prepped one colony each and digested them with EcoRI/XhoI. I also took out K112226, K112209, K112213 and K112216 which I put into Righty to amplify, and mini-prepped/digested them as well. K112226 and K112213 both still look wrong, so I'm going to try and find them on old stock plates and re-grow them. (see "molly digest 8-13", lane 2-8 are molly14 #3, molly19 #1, 226, ladder, 209, 213, 216)

I went ahead and started DBBS with molly19 #1 and molly14 #3 because both looked fine from the digestion map. I ran 4KB for PCR, zymoed and eluted with 90ul water, digested with BsaI, DpnI, and BamHI (Lefty) or BglII (Righty), zymoed again and eluted with 30ul water. Then, I took 1 ul of each and ligated, transformed into TG1, rescued and plated them on Spec.


I picked two colonies each for molly19 #1 and molly14 #3 in pBca1256. They are streaked on a Spec and a ACK plate, and are growing in culture tubes upstairs. Tomorrow, I need to mini-prep them.

I also decided to digest the parts that I still don't have to send out to iGEM in EcoRI/XhoI so I can digestion map them. It should be noted that K112216 was practically empty, and needs to be re-amplified. If they look reasonable, I'll send them out for sequencing.


I ligated 1ul each of molly19 #1 and molly14 #3 with pBca1256, and transformed them into TG1 cells which I plated after rescuing on a Spec plate. The first time around, I heatshocked for like, 6 minutes, so I started over again. All four are on the Spec plate. Tomorrow I need to pick/screen colonies.


Came in today and nothing grew on the plate. I guess it wasn't a colony I was seeing on Saturday. Jin told me to try the DBBS method to assemble my parts. So I set up two 25ul scale PCR reactions. One was with 0.25 ul of molly19 #1 reamp as template + JH111F + G00101, and the other was with 0.25 ul of molly14 #3 reamp as template + JH111R + ca998. I ran both with the 4K55 program. Then I realized that there's an AlwNI site in molly14... So scratch that.

I decided to transfer these parts back into pBca1256 and try the PCR method with those oligos since I know XhoI is specific to only one part of the plasmid. I digested molly19 #1 and molly14 #3 with EcoRI/BamHI and zymoed the samples. Tomorrow I need to ligate, transform, rescue and grow on a Spec plate.


I mini-prepped molly14 #3 that I transformed into TG1 cells yesterday, and digested 8ul with BglII/XhoI. I also digested 8ul of molly19 #1-1 with BamHI/XhoI, but it looks like I need to amplify this too. I'll transform it into TG1 cells later on today.

I cut out the bands for molly14 #3 and molly19 #1-1, zymoed, ligated, and transformed them (along with 1ul of molly19 #1-1). K112238 is plated on a CK plate and is in the warm room. molly19 #1-1 is in a culture tube in the lab's shaker. I need to mini-prep this tomorrow and hopefully pick colonies for K112238!


I digested the 5 colonies of K112234 in 1256 that I mini-prepped yesterday with BamHI/XhoI and ran them on a gel. See image "molly digest1 8-7" in which lane 1 is ladder, and lanes 2-6 are colonies 2-6. They all look great, so I'm sending out colony #3 for sequencing.

I am also trying to assemble {rbs.xis.int!}{rbs.ihfA!}{rbs.ihfB!} (K112237) with {b1006}{ccdB} (K112236) to make {rbs.xis.int!}{rbs.ihfA!}{rbs.ihfB!}{b1006}{ccdB} (K112238). I digested K112236 with BglII/XhoI and molly19 #1-1 and molly19 #2-1 with BamHI/XhoI. This gel showed no DNA! Ah! I again digested K112236 with BglII/XhoI and molly19 #1-1 with BamHI/XhoI (since I didn't have enough K112236 for molly19 #2-1 also). I gel purified and zymoed them, then ligated and transformed into TG1 cells.

I also transformed molly14 #3 into TG1 cells to amplify the product. I will have to mini-prep these tomorrow.


BBQ with UCSF!

I took out the 6 colonies that Bing picked yesterday of Lefty cells with K112234 in pBca1256. During mini-prepping, I accidentally added the colony 3 cells to the 1 cells in a 2 ml tube, so I don't have the colony 1 mini-prep. However, the rest are fine and are taped to the top of my box. :)


Museum Day!

I came back into lab and it turns out that the only co-transformation that I had was in molly19 #2, colony 3. So I mini-prepped the rest of them, and digested them with BamHI/XhoI. The gel (see image "molly digest 8-5") is as follows: Lanes 1-6 = molly19 #1 colony 1-6, Ladder, Lanes 8-12 = molly19#2 colony 1,2,4,5,6.

Bing also picked 6 colonies from the plate of Lefty cells with K112234 in pBca1256. (Thanks!) They are in the warm room and need to be mini-prepped and digestion-mapped tomorrow.


Jin looked at the digestion map from yesterday and said that the rbs.xis.int! from pAH57 in 1256 in Mach1 probably didn't work (since Mach1 cells were contaminated and would grow on Spec regardless of whether or not it has the plasmid in it) so I'm not going to send these out for sequencing. However, I am still sending out K112234 in CK #1 and #2 for sequencing with both forward and reverse oligos.

I looked at the 1-2-3 Assembly with K112234 in CK #1 and #2 with molly13 #2 into Lefty plate and there were actually colonies growing! I will let them grow for a bit longer, then pick them. At some point tomorrow I will need to mini-prep/digestion map them.

Jin also wants me to use my K112234 PCR product, ligate it into pBca1256, and transform into Lefty cells since this will be what Chris needs. They are on a Spec plate growing upstairs in the warm room.


I mini-prepped each of the 6 colonies for rbs.xis.int! from pAH57 in 1256 in Mach1 and for rbs.xis.int! from pAH57 in CK in Lefty. (Note: I eluted with 40 ul of water instead of 50 ul) All colonies grew on their stock plates. We discovered today that Mach1 is also contaminated with something that is resistant to Spec, so I will digestion map these samples and send them out for sequencing to make sure all is well with them.

I digested the two K112205 samples from yesterday with BamHI/XhoI along with the other 12 samples. I ran them out on a gel (see image "molly digest bam-xho 8-3", in which the first two lanes are K112205 #1 and #2, the next six are K112234 (rbs.xis.int!) in pBca1256 #1-6, then ladder, then K112234 in CK #1-6). The bands are REALLY light, but K112234 in CK looked relatively correct. I have no idea about the ones in 1256, but there seems to maybe be bands... Tomorrow, I will send out K112234 in pBca1256 #1 and #2, as well as K112234 in CK #1 and #2 for sequencing.

I went ahead and did 1-2-3 Assembly with K112234 in CK #1 and #2 with molly13 #2. Since the bands were so light, I used 2 ul of each plasmid. They were transformed into Lefty cells and are upstairs growing in the warm room on a CA plate. Tomorrow, I will hopefully be able to pick colonies.


I mini-prepped the K112205 rbs.xis! in 1256 in Righty once, but they zymo column looked kind of weird (like the filter had come up a little bit?) so I went ahead and mini-prepped it again. I have both in my box, and I will digestion map tomorrow.

I also picked 6 colonies each for rbs.xis.int! from pAH57 in 1256 in Mach1 and rbs.xis.int! from pAH57 in CK in Lefty. They are growing on stock plates and in culture tubes in the warm room upstairs. I need to mini-prep, digestion map, and send these out for sequencing tomorrow.


I took out the rbs.xis! (K112205) from Plate #3.1 C6 into righty which I plated on a Spec plate last night and there were many colonies. I picked one and put it in a culture tube with 2YT Spec. It's on the shaker in the warm room to mini-prep tomorrow and digestion map. Then I will ligate it with the rbs.int! that I gel purified yesterday.

I also ran my PCR product out on a gel, and it came out to be the perfect size (~1300bp), so I purified it from the gel via Zymo, digested it with Eco/Bam, did a zymo clean-up and eluted with 6 ul. I took 1 ul and ligated it into 1256 and 1 ul and ligated it into CK. I transformed the one in 1256 into Mach1 cells and CK into Lefty. They are plated and are in the warm room to grow overnight. Tomorrow I will come in, mini-prep rbs.xis! in 1256 in Righty, and pick colonies for rbs.xis.int! from pAH57 in 1256 in Mach1 and rbs.xis.int! from pAH57 in CK in Lefty.



Doesn't look like molly12 is working again... The plate I put in on 7/30 didn't grow any colonies, nor did the plate I put in on 7/29 and left in the incubator for 2 nights. Jin had be go back to the stock plates where rbs.xis! and rbs.int! were in the Spec 1256 plasmids. I digested 3 ul of rbs.xis! from Plate 3.1 C6 with BglII/XhoI, and 3 ul of rbs.int! from Plate #1 C4 with BamHI/XhoI for 1 hour in the 4th floor warm room. I ran the samples out on a gel. It seems like rbs.int! was the wrong thing in the stock plate (we saw a tiny band at about 300 and another single larger band at 2000, but no band at 1500). I will try digesting rbs.int! in Plate #3.1 C11. rbs.xis! was mostly single-cut, but the right bands MAY be there. I will transform the rest of the sample into righty cells and grow them over night to amplify the amount of sample we have.

I ran out rbs.int! and it looked like there were 2 bands, but both were a little bit higher than they should have been. Shit! I actually needed samples from Plate #1 C5 and Plate #3.1 C12! Ahh! Okay, so I am re-digesting 5 ul of each of these with BamHI/XhoI. They should run out to be about 2000 and 1500. I want to cut out the 2000 band and gel purify. Jin said they again both looked higher than they should be, but he cut out the larger band anyway and I purified it. It's on my box.

We also realized that both xis and int are in pAH57, so I amplified it with the forward oligo for rbs.xis! and the reverse oligo for int!. I put it in my box in the freezer.

Movie night!


I took out the plate of half TG1 and half Lefty transformed with molly14 #3, and there were quite a few colonies on the Lefty side, but there were CLEARLY more cells on the TG1 side. Since ccdB is relatively weak, there should be about 1/1000 Lefty cells that survived. There looks like there's more than that, but I think it's fair to conclude that ccdB is functional in molly14 #3. :)

My 1-2-3 Assembly with the new ig9 and molly2 #2 to make molly12 didn't seem to work. We think the DNA concentrations are too low, so Jin suggested I digest 8 ul each (w/1 ul NEB2 and 0.5 ul each of Bm/Xho or Bgl/Xho) and I will gel purify ig9 while just zymo-ing molly2 #2. I will elute each with 6 ul of water, add 2 ul ligase buffer, 0.5 ul ligase, and 5.5 ul of water.


Bing mini-prepped ig9 in the culture tube upstairs in the warm room (thanks!). I digested it with both BamHI/XhoI and BglII/XhoI and ran it out on a gel (see image "molly digest 7-29" in the first two lanes). The first lane is ig9 digested with BamHI/XhoI, and shows only a single band (i.e. single cut) and the second lane is ig9 digested with BglII/XhoI, and shows the predicted band lengths of about 1200 and 2450. :)

The transformation of molly14 #3 shows no colonies yet, but I just realized it's because I plated on [CK] instead of [AK]... damn! I did it over again, but accidentally added 42 ul of KCM to the Lefty cells instead of 30 ul, but hopefully they'll be okay...

I went on to do 1-2-3 Assembly with this new ig9 and molly2 #2 (1 ul of ig9, 1 ul of molly2, 1 ul NEB2/ATP, 0.3 ul each of XhoI, BamHI, and BglII). I then transformed them into Lefty cells left over from molly14 (see above), rescued and plated them.


Bing ended up doing most of the stuff I had on my list for Jin (thanks Bing!) the molly12 1-2-3 one and only colony turned out to be co-transformed... Sucks. It also turns out that the culture of ig9 was pink, but Bing mini-prepped it anyway... but the plated ig9 looked okay, so I picked a colony and put it into a culture tube with 2YT [AK] which is in the shaker upstairs. I threw away Bing's mini-prep per Jin's request. Tomorrow I will mini-prep and digestion map the picked colony, and hopefully successfully do the 1-2-3 method for assembly with molly2 #2. Today, I also made 2 more bottles of 2YT and poured out 2 24-well plates for Bing.

My transfer of K112229 Bm/Eco [AC] into Lefty from miniprep was sequenced and is correct! Hah! Enjoy Madhvi and Aron! :)

I also transformed molly14 #3 into both lefty and tg1 cells and plated them to make sure ccdB was functional.


Vacation from lab!


I took out the molly12 1-2-3 plate from the warm room and saw that it was still only 1 colony that was growing, so I picked it, streaked it and put it in a culture tube to grow up in the warm room overnight. I also took out the plate that was growing the K112229 Bm/Eco [AC] transformed into Lefty, picked 2 colonies, streaked and put them in culture tubes to grow up in the warm room overnight as well. Tomorrow, these should all be mini-prepped, digestion-mapped, and maybe sent out for sequencing.

I also took molly2 #2 and ig9 and digestion-mapped them in this order: molly2-2 Bam/Xho, molly2-2 Bgl/Xho, ladder, ig9 Bam/Xho, ig9 Bgl/Xho, bing's bx9 sample. See image 'molly digest 7-25.' I actually ended up cutting out the 2200 band in the first lane, and the 2500 band in the fourth lane, zymoed it to purify the DNA, ligated 7.5 ul with 1 ul water, 1 ul ligase buffer, and 0.5 ul dna ligase, and transformed this into lefty. I also transformed 1 ul of ig9 miniprep into righty because the digestion mapping suggested that the bam site is currently not methylated. ig9 was plated on an [AK] plate labeled "ig9 [AK] into Righty from miniprep," and it is also in a similarly labeled culture tube. Molly12 is on a plate labeled "molly12 into [CK] Lefty using gel purification." All plates are upstairs in the 4th floor warm room.


molly17 is perfect! Yay! However, the basic parts were wrong for rbs.lamB> and rbs.phoApp>, so it's useless... all 16 colonies that I picked for molly12 were also co-trasformed, as were all but 1 of the 8 colonies that I re-picked for molly14. I mini-prepped this, digestion mapped it (twice, second time using NEB2) and it actually looked good. I missed Richard for sequencing, so I'll send it out tomorrow.

I also digested Madhvi's ig1124 #1 sample (again, twice) and cut out the band, but I'll hold off on ligating it with the gel purified ccdB until I get the sequencing data back tomorrow. Both samples are in my plastic box in the freezer.

We had a mini-meeting today, and it was decided that the gateway reaction could not occur in the periplasm, so we're not doing my composite part with the prepro's anymore. I guess that's okay with me seeing as how 2 of the prepros are fucked up anywho.

We were having problems figuring out where rbs.pelB> was, so I took my old K112229 PCR product (which I think is actually rbs.pelB>) and ligated it with some of the digested AC vector and transformed it into Lefty. I plated it and it's up in the warm room growing.


Yay! All my sequencing data showed that molly13 #2, molly15 #1 and molly18 #5 are all correct! I shall throw away the second trial of molly18 since it worked out. Also, for assembly, five of the 6 colonies picked for molly17 were not co-transformed, so I mini-prepped and digestion-mapped them with BamHI and XhoI (see image molly17 digest 7-23). All looked fairly good, but molly17 #5 had the brightest bands, so I will send that one in for sequencing.

Of the plates that I put back into the incubator overnight, more/smaller colonies grew on molly12 and there were some more molly14. I repicked 16 for molly12, and 8 for molly14 and they are growing upstairs on plates and in a 24-well block named Ricky. Hopefully at least 1/16 will work... However, just in case, I will be doing the 1-2-3 Assembly of molly12 (molly2 and ig9) and I will be re-assembling molly14 but digesting/cutting out of a gel. Molly12 is plated and is upstairs in the warm room.

So... molly3 (b1006 in AC that Madhvi gave me, aka ig1124) didn't separate into the appropriate bands of 1251 and 1999. I think Madhvi is getting b1006 sequenced. I cut out the 2900 bp band which is molly4 digested with BglII and XhoI, and Zymoed it. It's in my box labeled "molly4 gel purif. Bgl/Xho." Hopefully I'll be able to gel purify molly3 and ligate them together so that I can greatly reduce the rate of co-transformation in molly14.

As for molly8 and molly9 that I transformed into lefty/righty, I will hold off on assembling them using the 1-2-3 method until I get my sequencing data back tomorrow since molly17 looked pretty good when digestion mapped.


Today is not starting out very well. I got sequencing data, and for molly6 it was perfect, but for molly7... it turns out that rbs.pelB> is actually a~pelB> on the stock plate, so both Aron and I have to start over with that piece.

Chris came in and said that we need to incubate for longer before we pick colonies. This might lessen the frequency of co-transformation. Therefore, I will put all the 7/20 plates back in and see if more/smaller colonies grow.

In Fred and Ethyl, molly15 #1 and #5, molly18 #3-5, and molly13 #1-8 all worked fine. I accidentally tube-switched molly13 #7/8, so I didn't bother with those. So I mini-prepped molly15 #1 and #5, molly18 #3-5, and molly13 #1-3, and digestion-mapped them (see image molly digest 7-22). All but molly13 #1 looked fine, so I sent out molly13 #2, molly15 #1, and molly18 #5 for sequencing.

Jin also wanted me to re-transform molly8, ig9, molly2, and molly 9 in case molly12 and molly17 don't turn out well. They are upstairs in the shaker in culture tubes.

I also picked 8 colonies for molly14 and molly18 (and all 6 for molly17) and streaked them/grew them. Hopefully they will all turn out fine.


So molly15 and molly12 both had a lot of colonies, while molly13 and molly16 had a fair amount (around 15?). However, molly17 and molly18 had very few/no colonies, so I will have to do these over again. Jin thinks that maybe they're toxic to the cells, but it might also just be that the DNA wasn't concentrated enough. I'll try it again and replace the water with more plasmid.

I picked a lot of colonies for the other assemblies that did work. They are on plates and in 24-well blocks labeled Fred and Ethyl.

I'm doing assembly with Madhvi's b1006 [AC], clone #3. I will call it molly3, and will assemble it with molly4 to make molly14. I am doing assembly over again on molly17 and molly18, this time using clone#2 for each.


Jin looked at my gel for ccdB (image molly bm-xho 7-19) and said that even though the bands are light, they are where they're supposed to be. Thus, I think I have all my transfers ready to go (aside from molly3 which Madhvi still has to mini-prep and restriction map digest).

I started assembly today. I plan on doing molly12,13,15-18. I'm skipping molly 14 because I still don't have the terminator that Madhvi is making.


Jin looked at the gel I ran last night and said they all looked okay, but to sequence molly6 (<int! in K/A). Aron also mentioned that his rbs.pelB> part was actually a~pelB>, so we need to figure out which stock plate was switched. Thus, I am also sending in molly7 (rbs.pelB> in AC) for sequencing. If it is also a~pelB>, then there's something wrong with the original stock plate.

I found that only ccdB colony #1 and ccdB colony #3 were okay (ccdB colony #2 grew on Spec). I also screened Bing's 5 samples and spun down the samples that didn't grow on the Spec plate and put them in his box. I mini-prepped ccdB #1 and ccdB #3 and digested 5 ul each with BamHI and XhoI (3 ul water, 1 ul NEB3, 0.5 enzymes).


I checked the samples upstairs and it turns out that molly4 (ccdB) actually grew this time in the TG1 cells (yay!) so I picked 3 colonies, streaked on CK and Spec plates and grew them up in a 24-well block with 2YT CK. I did this along with 5 of Bing's samples, and so they are all upstairs in the warm room. The 24-well block is named Jesus and is on the shaker.

I also looked at the plates that I screened for yesterday (molly2,5-11) and got 3 viable colonies for each molly10,9,7,11,8. Colonies 1 and 3 of molly6 grew on the spec plate, as did colony 1 from each molly5 and molly2. After spinning down the cells during mini-prep, I discovered that colony 3 of molly5 was pink, so I discarded this one as well. I mini-prepped the other 19 samples and digested 5 ul of each with BglII and XhoI to do digestion mapping. It looked like there was something wrong with the enzymes because there were A LOT of single cuts. However, we could see that molly9 and molly11 were okay. See picture "molly_bglII_xhoI 7-18 2". I'm digesting all the rest with BamHI and XhoI, so hopefully I'll get better results. See picture "molly bamhi xhoI 7-18"


Clonies grew on molly2,5-11 but I realized that molly4 (ccdB) is actually toxic to the righty cells... so I am transferring it again today using TGI cells (w/F plasmid). I picked 3 colonies each for molly2,5-11, streaked them on double-antibiotic stock plates, on a spec plate, and grew them in a 24-well block. Tomorrow, I need to screen them to make sure at least one colony grew on the double-antibiotic plate and didn't grow on the Spec plate. Then, I need to mini-prep all of those samples, digestion map them and hopefully start on assembly.

Today I mini-prepped and digestion mapped ig9, ig28, ig26 and ig122 with BamHI and XhoI. They all looked good. :)


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. They were transformed into righty and lefty cells and are on 3 plates growing in the warm room. Tomorrow afternoon, I will pick/grow them up.


Didn't come into lab today.


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.


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.


Kayaking trip! :)


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.


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.


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.


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.


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.


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!)


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.


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.



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.


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.


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!


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.


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.


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.


Madhvi picked colinies for K112200, K112201, K112204, and K112205 for me. (Thanks!)


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.


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!)


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.


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).


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.


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.


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.


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.