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Team:UChicago/Notebook
From 2014.igem.org
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==Week 10 8/18 - 8/22== | ==Week 10 8/18 - 8/22== | ||
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| + | ==Week 11 8/25 - 8/29== | ||
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| + | ==Week 12 9/1 - 9/5== | ||
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| + | ==Week 13 9/8 - 9/12== | ||
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| + | ==Week 14 9/15 - 9/19== | ||
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| + | ==Week 15 9/22 - 9/26== | ||
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Revision as of 04:29, 17 October 2014
You should make use of the calendar feature on the wiki and start a lab notebook. This may be looked at by the judges to see how your work progressed throughout the summer. It is a very useful organizational tool as well.
This needs to get split up into separate pages somehow
See Media:UChicago_LabNotebookSummary.pdf for the original document.
Contents |
Week 1 6/16 - 6/20
Monday
First day of lab! We plated some DH5-alpha competent cells in preparation for gDNA extraction for our PCRs and future transformations. One person showed up (Kevin yayyyyy), then left.
Tuesday
We attempted to make LB plates. We had fun watching it boiling over since we left it in the hot plate too long and it spilled everywhere. We did clean-up time afterwards! In the future, we will remember to to heat for only 1 min to dissolve everything except agar. Agar will dissolve during autoclave because science is cool. So exciting! :D We then remade the LB for funsies :D We did a few other things to start things up like making SOB, CCMB80 competent cell buffer, and antibiotic plates.
Wednesday
MOAR BUFFERS! Qiagen buffers are expensive so we spent the day making bootleg Qiagen miniprep buffers (sorry Qiagen, you had your chance). Then, we contemplated life for the rest of the day. *take depressing pictures here*
- take pictures of buffers*
Thursday
Made competent cells. Stuck hands in liquid nitrogen. Burnt our hands off (no pics because you need hands to use a camera, duh). EVEN MOAR BUFFERS We also went to a biotech company exhibit and grabbed a lot of free samples! (and food of course) We also extracted gDNA from DH5a to PCR out our mutator genes. We also learned not to use the free 1.5mL centrifuge tubes from last year, and to not smell our microcentrifuge anymore. Stock strains MG1655 and delta-tyrR came in from the Yale E Coli Stock Center. Yay! So exciting! Kevin was so excited that he burnt his hair trying to plate them. XD
We made competent cells (MY CRYSTAL BALL SAYS: THEY WILL FAIL). Kevin BS’d the gDNA and got some somehow but also made the centrifuge stink like chloroform/phenol. Fun XD
This company (name concealed) gave us some cool centrifuge tubes last year that warp when we spin them down. XD So we’re buying new ones. $$$ They’re wrong- money DOES grows on trees! AND we have lots of trees around here! Even better! We ran overnight PCRs, one with more and one with less cycles.
Friday
PCR-ed out 9/12 mutators. The lengths of almost all of them were verified to be correct!!! So much happy!!! Yes!!!! Yay!!!! The PCR with less cycles (bottom half of the gel) worked better so we’ll stick with that in the future. We also digested backbones that have the RBS we will use and ligate to all the mutators. What a good day!
Week 2 6/23 - 6/27
Monday
We tested our competent cells using the transformation efficiency kit. They didn’t work XD But that’s okay, we like making competent cells. It’s strangely exciting to pipette things lots of times :D
Also, we fragment isolated the mutations and then did the restriction digests on the PCR products. The concentrations of the genes are fairly alright, about 20-50 ng/microL. We ran a gel of AroF to verify PCR of the gene, but unfortunately we did not get a band XD. That’s okay, life goes on even if your gels fail!!! XD IT’S summer time! Rainbows!
We gel extracted the mutators that were digested last week. Fun. Got high contamination. But contamination is good because what doesn’t kill your experiments makes them stronger right ? Yes! Concentrations are listed below XD
| Sample | Conc (ng/microL) | 260/280 | 260/230 |
|---|---|---|---|
| ParC | 30.2 | 1.78 | 0.25 |
| DinB | 55.8 | 1.70 | 0.42 |
| MutS | 21.3 | 1.86 | 0.18 |
| Dam | 37.0 | 1.68 | 0.18 |
| MutT | 29.9 | 1.61 | 0.09 |
| Umu’C | 24.5 | 1.80 | 0.05 |
| EmrR | 36.8 | 1.71 | 0.08 |
| DnaE | 31.1 | 1.53 | 0.32 |
Tuesday
NOTHING WORKED :)))))!!!!!!!! We drank our worries away XD XD XD. We drunkenly made glycerol stocks of the two strains from Yale that came last Thursday.
- hic* guuysh guysh we should *burrrrp* totally ligate a rBs to a vecter *hi*c
Alcohol is poison. (Delicious, delicious poison. And we are Mithridates.)
Unfortunately, our cells weren’t competent when we tested them again :( We went over the protocol and made sure we’ll have everything down for next time XD. Or rather, fortunately! We get to make more! XD Soooo stoked! We fragment isolated and gel purified our promoter plasmids. For our project planning, we met with our faculty advisors Mike and Jennifer. The meeting was super, super, super productive and educational! We could consider inserting a polylinker into our plasmids to reduce a lot of steps XD Polylinkers are fun!
Wednesday
We purified the 8 mutators that we PCR-ed out. As for AroF and the three mutators that didn’t work, we tried PCR with Phusion, but unfortunately it didn’t work again. Yay! After digesting our RBS and ligating it with our previous cut promoter plasmids, we also did 3A assembly in parallel for RBS and promoter. Yay! More amp and tet plates were made! Yayyy!
- picture of plates”
Thursday
We had issues with SOC contamination today - We had to make a new batch for more transformations. We also tried transformation with the efficiency kit again, making sure we were following each step as it was written on our protocol. Friday Transformation of our 3A assembly colonies didn’t work, but the transformation of mcherry plasmids worked! This means that there’s nothing wrong with our transformation protocol. We will look into our digestion and ligation steps. We <3 troubleshooting! All part of the scientific process XD! Here’s a joke XD: What do you call it when McDonald sells cherries? mCherry! HAHAHA XD!
We made more chloramphenicol plates again! Yay!
Week 3 6/30 - 7/4
Monday
We resuspended and transformed the promoter+RBS plasmids from the distribution kit. They turn red when resuspended! Such a pretty color!
Tuesday
A lot of our reagents came in today! WOOOOOOOOo! Since we’re having problems with transformation right now, we are troubleshooting our procedures to see if there’s anything wrong with our technique or protocols. We followed the iGEM standard transformation protocol and transformed our promoter+RBS plasmids again. Let’s hope they work!
Week 5 7/14 - 7/18
Monday
We got the plasmids we ordered last week, including the 2 melA constructs (K193601 and K193602). We might not need them for a while, but always good to have them on hand. They came in agar stabs, so we restreaked them on plates. A bunch of our media was contaminated (again) so we had to remake all those. We also transformed our IPTG+GFP and our AroF+mCherry constructs, along with Cam, Kan, Tet, and Amp linearized backbones from the kit plate (other plasmids, cut in order to isolate the backbone).
(I’m calling this transformation.jpg now.)
Tuesday
Yep. Transformation of our ligated parts and the backbones seems to have failed. We left the plates in the incubator for one more night because these cells have an annoying habit of only growing after two days. At least our stab streaks were good: all 7 of the plasmids, including the important melA constructs, grew successfully, and were made into overnight cultures. Oh hey! After a few hours more there is some growth on the backbone plates, except for the Tet backbone. Transformation efficiencies are pretty terrible, as is par for the course here. Seriously need to fix that. We also redid our AroF+mCherry and our IPTG+GFP ligations with 3A assembly and the standard ligation procedure. Better work this time. We also miniprepped and fragment isolated more mCherry and GFP so that we have enough for the inevitable future redos. We’re also continuing work on determining the MIC for rifampicin for our fluctuation assays; this time growing them in liquid culture.
Wednesday
Made glycerol stocks of most of Monday’s new plasmids, and miniprepped K193601 and K193602. Ran a bunch of PCRs to make sure we had our promoters. Didn’t get IPTG+GFP, but at least we got a little bit of AroF+mCherry. We also checked our MIC liquid cultures, and it looks like the MIC we got was about 8ug/mL. We also transformed our ligation products into our competent cells and competent cells from another lab. The other lab’s cells will most assuredly have better transformation efficiencies than our cells.
Thursday
Looks like our 3A fragments were cut incorrectly due to mislabeling somewhere along the line. Would probably explain why they failed this morning. We can make our competent cells better, more competent. We have the technology. And buffer. Which we made today.
Friday
Plated some XL1-Blue cells to start making our competent cells. Hopefully these ones work better. Also tried ligating and transforming more mutators and promoters; hopefully we can do this without mislabeling things again. (ipredictfailure.jpg)
Saturday
Made a culture of XL1-Blue cells with 250mL LB medium, growing cells to an optimal OD of 0.6 to transform. Looks like this will be going overnight, as growth is slow. Yesterday’s transformation products finally worked without incident: making them into overnight cultures so that we can have stocks in case of temporary incompetence.
Week 6 7/21 - 7/25
Monday
We are spending the week attempting to clone our mutators into our IPTG-inducible promoter+GFP plasmid. We used up some of the fragment isolated mutators we had, so we gel purified more from wild type. The mutators and IPTG+GFP were appropriately digested (mutators with XbaI and PstI and IPTG+GFP with SpeI and PstI). We also performed a cell competency test on our cells (which are probably not competent, knowing our luck).
Tuesday
At the suggestion of our advisors, we are now dephosphorylating our plasmids before cloning to decrease background (which was a problem with our previous digestions). This should help a lot yay!! We dephosphorylated IPTG+GFP/S+P and gel purified the dephosphorylated digest. We cloned the mutators into IPTG+GFP using a ratio of 3:1 insert:vector in a 10uL reaction with 25ug of vector. We also submitted IPTG+GFP, mCherry+AroF, promoter+TyrR for sequencing. (We also made a ton of cam plates so no one freaks out the next time they’re transforming something and realizes there are no plates). This was a super late day--at lab past midnight=fun, fun, fun :)
Wednesday
Our SOB is contaminated and an aliquot of SOC that was never used before contaminated. What is this madness?!?! More SOB was made. We also colony PCRed IPTG+GFP, mCherry+AroF, and promoter+TyrR in preparation for negative sequencing results (Always be prepared is one of our many mottos. We stole it from the Boy Scouts.). We also got Tyrosine!!!!
Thursday
Well, our sequencing results came back, and it appears we have MAJOR issues with mislabelling. While we do have IPTG+GFP confirmed, aroF and TyrR are not in anything The plasmid mcherry is not even in anything. What was labeled as aroF+mCherry actually contains the general promoter, and one labeled promoter+TyrR is actually IPTG but without GFP!!! Are our mCherry and promoter plasmids mislabeled?! Or was it just the ligation products? The struggle is five real. We made overnight cultures of all the ligation products that looked promising based on the amount of colonies (our colony PCR did not work and this will allow us to keep moving forward while troubleshooting colony PCR): 4x IGMD (IPTG---GFP---MutD), 4x IGMH (IPTG---GFP---MutH). 4x IGMT (IPTG---GFP---MutT). 4x IGDam/IGD (IPTG---GFP---Dam). 4x IGUC (IPTG---GFP---Umu’C), 4x IGER (IPTG---GFP---EmrR).
Friday
Miniprepped all overnight cultures that were made yesterday of ligation constructs. We then PCRed them and chose constructs to submit for sequencing.
Week 7 7/28 - 8/1
Monday
This week we’re continuing lots of cloning. We were planning to make competent cells, but messed up the OD (which should be about 0.6) :(. Sadness. PCR’d out the AroF promoter and the TyrR gene and fragment isolated them.
Tuesday
Re-inoculated new cells to retry making competent cells again. Hopefully they work this time. Did appropriate digestions on paroF, TyrR, the mCherry plasmid, and the IPTG-GFP mutator plasmid as we continue to clone all three different constructs (mutators, general promoter-TyrR, and paroF-mCherry). Did PCR’s using VF and VR2 primers to confirm the identity of the mCherry and constitutive promoter plasmids. However, mCherry looks possibly bad, so we ended up going ahead with cloning of only TyrR into the constitutive promoter plasmid. We got back sequencing results for three previous attempted ligations of Dam, EmrR, and MutT into IPTG-GFP, all of which look good. Yay, first real biobricks!
Wednesday
The cell line we’re using to make competent cells (XL1-Blu) that we’re trying to grow up is growing up faster than we expect, so we made a growth curve for how the OD changes over time. Did colony PCRs on attempted ligations of various mutators (Mut L, MutS, DinB, DnaE, MutY) into TyrR, but they all looked bad. Ligation fail.
Thursday
We made competent cells today finally, and transformed some test plates to make sure they actually work. We redid the PCR to verify the mCherry plasmid, which again looked bad, so we’re just going to retransform mCherry just to be safe.
Friday
Success! Competent cells are actually competent after about 12 hours of growth. They look possibly pink, just as expected (since the plasmid they’re transformed with expresses RFP)
Week 8 8/4 - 8/8
Monday
Resuspended and transformed mcherry+lva from the distribution kit again. We've been using this a lot and looks like we’re running pretty low on this! Let’s hope the transformation will succeed and our glycerol stock will be fine! Also transformed the sequence verified IPTG+GFP plasmid and those with EmrR, Dam and MutT for later use in the mutation rate analysis.
Tuesday
Transformation yesterday seems ok! Since there are a few colonies on the negative control, we restreaked the transformed colonies just to be sure. And finally, the long-awaited MIC determination test was done today! As a preliminary experiment, we plated saturated, 1:100 and 1:10000 dilutions of XL1-Blue cells onto cam plates with various rif concentrations.
Wednesday
The restreaked colonies grew! Promptly checked the colonies by colony PCR and made overnight cultures of them for miniprepping.
Only less than 5 colonies grew on the 5ug/ml rif plates, and none on the other plates! This is unexpected, as there should be as many colonies as the plain LB plates in the lower rif concentration plates. Looks like there’s gonna be more rif experiments!
Thursday
As we accidentally added an extra RBS overhang in our AroF primer, we PCRed out AroF again using our new primer without the overhang. The gel looks good! Let's miniprep it!
And what??? 4 out of 7 overnight cultures from yesterday didn’t grow? And we have to pick the colonies again? We really need mcherry+lva… :( But the IPTG+GFP plasmids look promising. Miniprepped the plasmids and ran them on a gel. Look at this beautiful gel! Now let’s cut it up for gel extraction!
Friday
What? Why didn’t the mcherry colonies grow again? :( Retransformed mcherry again. Ligated promoter and TyrR while waiting for transformation. For the mutator plasmids, the remaining mutators were digested and ligated into the IPTG+GFP plasmids. Transformation results will be ready tomorrow! The ligated MutT and EmrR plasmids from earlier this week were miniprepped and stored.
Week 9 8/11 - 8/15
Monday
It looks like one of the tubes labelled as “promoter plasmid” was actually IPTG+GFP. Whoopsy-daisy. Also transformed general promoter from kit plate. A lot of the mutator transformation plates worked. Woot! We can now use them for cloning and stuff. Did colony PCR for a bunch of them and then ran on gels. MutH, MutY, and UmuC all gave good results.
Tuesday
Made glycerol stocks of mCherry. Miniprepped mCherry+Iva, then restriction digested and gel purified it. Ran colony PCR reactions for MutS, MutL, and DinB. Ran gels for DnaE, UmuC (redo), and ParC (redo). The redos were done because redundancy is useful in science. Redundancy is redundant. Redundancy is also redundant. Made a bunch of variable Rifampicin plates and diluted MG1655 into 200ul aliquots for mic determination.
Somewhere, in a deep, dark corner of the gel room, someone started making new TAE Buffer...but their mind was in physics mode instead of bio mode…*foreshadowing intensifies*
Wednesday
Did colony PCR on a boatload (okay, it would have to be a really small boat) of IPTG+GFP so that we’d have plenty for future gels. Ran gels for MutS, MutL, and DinB. MutS and DinB gave some good results, but the two MutL gels didn’t work (one showed nothing, the other disintegrated). And so the buffer problems began. Miniprepped MutH, ParC, UmuC, DnaE, and MutY. Twice. The first time failed miserably, since it turns out that one of the buffers (P2) was improperly made and had been giving us screwy results for a while. Some members wanted to keep it for some reason, so we just gave it a choke collar label that said “BAD” on it. The second miniprep attempt was successful with a better-behaved buffer. Made glycerol stocks of promoter. Restriction digested and gel purified promoter. Ligated mCherry+paroF and promoter+TyrR and transformed products. Plated independent cultures of MG1655 onto restrictive media and made dilutions (only up to around 10^6, though, nothing like that 1/10^30 homeopathy BS)
Thursday
Sent in a variety of mutators (MH, MY, UC, DE, and PC) for sequencing. Miniprepped DinB and MutS samples. Digested and ligated MutL and MutD with increased insert:vector ratio (now 5:1). Did Colony PCR on IGER3 and IG3 samples then ran on gels (used separate TAE buffer from the main stock, but the gels still failed anyways).
Ran Colony PCR and gels for mCherry+paroF and Promoter+TyrR Nevermind! The buffer is DEFINITELY bad, so there go the results for that! Time to do it all over again *eye twitches*.
Made more LB, rif, and dilution plates since the results from the previous ones were inconclusive.
Friday
Re-did the gels for mCherry+paroF and Promoter+TyrR. Sent in DinB and MutS for sequencing. Transformed MutL, MutD, and IPTG+GFP into competent cells. It turns out that we didn’t need the IGER3 and IG3 overnight cultures that were made on Thursday, so they were thrown out. Made new LB agar plates. Selective growth plates from the other day showed no growth, but the nonselective test plates showed reasonable amounts of growth.
