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{{Team:UChicago/NavBar}} ===Day 1: 6/16/2014=== KY Plated some DH5-alpha competent cells in preparation for gDNA extraction and future transformations ===Day 2: 6/17/2014=== EJ, RK Made LB Agar for plates- 3 Liters *The solution mix was overheated and flowed over. Remember to heat for only 1 min to dissolve everything except agar. Agar will dissolve during autoclave *Annie has edited the protocol *Also remember to autoclave some stir bars so antibiotics can be added while mixing on stir plate afterwards. KY, RK *Made LB again **1 L *Replated DH5-alpha cells, they’re in the 37 degree heater *Inoculated DH5-alpha cells for gDNA prep tomorrow (possibly boil prep?), they’re in shaker. AZ *Made 1L SOB for preparing competent cells, with Kevin helping to adjust pH: **Protocol: http://parts.igem.org/SOB **For 1L, **#5 g yeast extract **#20 g tryptone **#0.584 g NaCl **#0.186 g KCl **#2.4 g MgSO4 *Made CCMB80 buffer for preparing competent cells, with Rikki helping to adjust pH: **Protocol: http://parts.igem.org/Help:Protocols/Competent_Cells **CCMB80 was stored in the 4 degree cold room. EJ, RK *20 chloramphenicol plates made *20 plain agar plates made *19 Kanamycin plates made AZ *Wrapped the plates in parafilm *Stored in cold room behind centrifuges - not sure if we can store them there. Someone ask Rust lab about 4C storage *KanR plates were not labelled with green. Realized after wrapping so labeled green on parafilm. Things we’ve borrowed: *Rust lab *Tryptone (~50 g) *Pipette tips (1 box of 1000 uL tips) *1 bag of 14 mL plastic conical tubes *1 box of 15 mL conicals *Reagents to make 1 L of SOB (~ 20 g tryptone, 2.4 g MgSO4, .186 g NaCl) *Reagents to make 1 L CCMB 80 *1 syringe and syringe filter *Aluminum foil *Weigh boats *Jennifer **1 mL SOC **LB kanamycin (~15 mL) **Inoculating tubes KY Plans *Need to grow up plasmids from iGEM distro kit tomorrow once plates are ready *Need to inoculate cells overnight *Need to replate cells *Make electrocompetency buffer. *Other buffers: I’ll make a list/figure out how to do everything tonight. *do gDNA extraction prep, PCR everything, run gels *start restriction digests (if time permits/everything looks good) Summary *Made plates with different antibiotics for future use *Replated and inoculated DH5-alpha cells for gDNA prep *Made buffers for competent cell preparation. ===Day 3: 6/18/14=== EJ KY *Made 50ml 0.5M EDTA solution, pH 8.0, filtered *Made 50 mL 10% SDS solution *Made 10 mL 1% NaCl solution RK *Made, filtered 250mL 1M Tris-HCl solution, pH 7.98 *Made, filtered 80mL 3M sodium acetate solution, pH 5.2 *New document in Reagents folder: cross-checked reagents,equipment we need with Gordon/Searle catalog (buy stuff tomorrow) *Resuspended 6 vectors from iGEM: iGEM 2014 wellplate 4 well 17O accidentally resuspended. iGEM 2013 wellplate 1 well 3M might have too much H2O, too dilute, *hopefully during transformation) EJ KY RK *Inoculated 3 tubes (see Kevin’s post below) RK EJ *Resuspended primers *Vortexed primers *Primers have been put in “-20” fridge. KY *I forgot that the shaker from Rust lab was being adjusted to a lower temperature overnight, so there wasn’t much bacteria this afternoon :/. (Note: This means you should check the schedule that’s taped to the shaker before you put stuff in it to make sure the temperature will be OK and not be stupid like me) *We’re re-inoculating 3 tubes for today: 2 for making electrocompetent cells and 1 for the gDNA prep which has be en postponed to tomorrow. We’ll also do all the PCRs tomorrow and start the digestions/cloning if they look good (else rerun PCRs under different conditions for any that don’t look good). *We’re also inoculating the RBS-containing plasmid today so we can get miniprep tomorrow. *We’ll inoculate the starter culture tubes for electrocompetent cells into 250 mL media overnight to grow for the day after tomorrow (? actually not sure: protocol says to do it at 20 degrees C but we can probably do it at 37 degrees C which means it would grow a lot faster) *Made SOC, transformed the following 6 vectors from the iGEM kit plates (with chloramp and amp negative controls of course): **RBS 34 plasmid (BBa_B0034): 2014 Kit Plate 4, Well 1N on amp backbone **RBS 32 plasmid (BBa_B0032): 2014 Kit Plate 2, Well 2J on chloramphenicol backbone; 2014 Kit Plate 4, Well 1J on amp backbone **RBS34+T7 promoter plasmid (BBa_K525998): 2013 Kit Plate 1, Well 3M on amp backbone **Generic promoter that we prefer to use (BBa_J23119) (labeled with a P): 2014 Kit Plate 3, Well 17O on chloramp backbone; 2014 Kit Plate 4, Well 17B on amp backbone Things we borrowed: *Rust lab *9.30 g EDTA borrowed *5 g SDS borrowed *~30 g Trizma base *~4-5 mL HCl for pH adjustment *1 250 mL filter *~12.3 g anhydrous sodium acetate *~2 50mL syringes *~2 syringe filters *~36 g glucose *5 amp plates *~8 tubes of 100 uL competent cells *Enough glass beads (~5-6 on average per plate) to spread 8 plates Things I will be borrowing tomorrow: *2 mL 1:1 phenol:chloroform *Need to remember to sterile filter CCMB80 *Need to check if we have 6X loading dye *Need to check what bp ladder sizes we have *Check parafilm? Summary *Made solutions and buffers to be used in later procedures *Re-inoculated three tubes, two for electrocompetent cells and one for the gDNA prep *Transformed general primer and RBS vectors from the iGEM plates *Readied primers for use later ===Day 4: 06/19/14=== RK *Filtered CCMB80 (in 4 freezer) *Added 600 microL phenol:chloroform:IAA (25:24:1) to tube with E. coli DNA *Filtered isopropanol *Made chloramp stock solution (25mg/microL) (excess is in “-20” freezer) *Filtered amp stock solution (100mg/mL) (excess in “-20” freezer) KY *The stock strains MG1655 and the delta-TyrR knockout came this morning. I put the filters in ~200 uL LB and streaked the plates. However, I didn’t really know what I was doing and burned my hair so hopefully there’s not contamination lol. KY,EJ,RK *1.5mL of overnight culture were pelleted, treated with 600mL lysis buffer, then incubated at 37 C for 1 hour. Removed at 11:05. *Treating lysed pellet with phenol/chloroform as per the protocol for gDNA prep on the protocol masterlist. White layer formed: may be the protein layer mentioned in the protocol. Spinning at 13.2 for 5 minutes. **White layer removed into another tube. Aqueous layer spun again with same parameters. **Adding another 300uL of phenol/chloroform. *Another 300uL of chloroform added to white layer. Spun for 5 minutes. *Multiple cycles of spinning and chloroforming. **May not actually have worked: spinning at 4 C for 15 minutes at 15000 to confirm. **Did not work *Second time around it worked (we are making changes to the protocol based on the amount of times we had to add phenol chloroform and centrifuge). (KY note: well possibly works, the 260/280 and 260/230 values are somewhat off and the thing smells like chloroform but visually the peak looks normal. Will have to do the PCR and see what happens.) RK, EJ *Received 300mg of amp from Goldbio person at GCIS science goods fair thing: preparing 3 1ml aliquots of stock solution as per protocol (100mg/ml conc). We have some amp left over (The bottle says 300 mg but we have 0.11 g left over). EJ *Inoculated 5 tubes with yesterday’s plated bacteria: 3 w/ amp and 2 w/ cam. Currently incubating. (KY Note: One of the transformations from yesterday didn’t work, the one with the RBS34 and T7 promoter, so I guess we’ll just have to do without it and stick with the original plan) *Inoculated with amp before it was filtered. (Remember to filter first in future) *Also inoculated another DH5a tube, currently incubating RK, EJ *Preparing competent cells: Seed stock at ~0.3OD after 5 hours. *Transferred to flat-bottom centrifuge tubes, spinning at 4 C, 3000g for 10 minutes. *Resuspended in 80mL CCMB80 *Distributed about 22.5 mL in eight tubes *Ice bath for 20 min *Centrifuge at 3000g, 4 C for 10 minutes *Resuspend in 10 mL CCMB80/4 tubes *RK--I messed up and did 10 mL CCMB80/2 tubes.We’ll check OD to see if it’s not too bad. If it is, I will re-chill, *centrifuge, decant, suspend half of the competent cells. RK *Made blank solution for measuring OD. Measured OD of my half (must do over, see above) and Eliots. *Elliot’s **OD=1.82 **Adding a bit CCMB80 to lower OD **Approx. 2ml CCMB80 added to cells **New OD= 1.36 *Rikki’s **OD=1.12 **Aliquoted into 80microL Stored competent cells in -80 freezer *Rikki: (Two batches: smaller first batch done w/o liquid nitrogen *second large box done with liquid nitrogen) *Eliot: One batch *Total yield should be around 125 tubes. KY *Got the (questionable) gDNA on the second try, so now we’re gonna try the PCR *Since I’m not totally sure how these primers will do, I’m gonna try 2 different cycling protocols (see masterlist, the one that works less well will be deleted tomorrow) *Here’s the master mix (27X for 11 genes): **135 uL 10X buffer **27 uL dNTPs ***uL 10 uM primer stock **27 uL DNA stock **13.5 uL Taq **1120.5 uL H2O **1323 uL total -> 49 uL per tube, add 1 uL 10 uM primer stock *PCR tubes: **1-12 = Cycling 1 (with 15 second cycles- see protocols for details) **13-24 = Cycling 2 (with 30 second cycles) **1- negative control, 2-mutY, 3-ParC, 4-DinB, 5-MutS, 6-Dam, 8-Umu’C, 7-MutT, 9- EmrR, 10- DnaE, 11- MutL, 12- MutH same, repeat for 13-24 **13- negative control, 14-mutY, 15-ParC, 16-DinB, 17-MutS, 18-Dam, 19-Umu’C, 20-MutT, 21-emrR, 22-DnaE, 23- MutL, 24- MutH If you need to centrifuge: use specially-marked bag of tubes (bag labeled CENTRIFUGE). The other tubes are pretty crappy and warped when Eliot tried using them in the centrifuge. (KY Note: They also leak when spun down and now there’s phenol-chloroform in the centrifuge so make sure you use gloves when you use the centrifuge) We have a lot of the poor tubes and not that much of the good ones, so we might need to buy more. $$$ cha-ching-cha-ching-cha-ching $$$ Things we borrowed: *60 uL of (Qiagen) protease *2 * 1000mL filter from Rust Lab *~10 mL of 1:1 phenol/chloroform *~5 mL chloroform *1 syringe filter *1 5 mL syringe *25 mL pipette things *another box of P200 tips Misc things we need: *Pipette tips *Eppendorf tubes (the ones we have are junk) *Pipette tip container’ *25 mL pipette things *PCR rack would be nice FOUND: *5 mL syringes *30 mL syringes *A complete miniprep kit not missing buffers Things we need to do tomorrow (just a reminder for myself): *Test competent cells *Do minipreps on the cultures we inoculated to get all the plasmids we want *Run PCR on gel to verify *Do restriction digests both on appropriate plasmids and on PCR products *Do ligation *Transform overnight *Design primers for the silent mutations and the TyrR MG1655 knockout *Replate MG1655 and the TyrR knockout *if PCR doesn’t work, try more downstream purification of DNA (do an additional ethanol precipitation in a binding column that I think we have) (http://www.protocol-online.org/biology-forums/posts/37302.html) -> also, EtOH precipitation appears better than isoprop precip (http://www.protocol-online.org/biology-forums/posts/3874.html) ===Day 5: 06/20/14=== AZ *Put NEB dNTP, ligase Promega GoTaq sample into freezer *Where is the *Autoclaved some eppendorf tubes, recovered from autoclave EJ *Running competency test on transformed bacteria made yesterday: 3 samples, one from each batch (Eliot’s, Rikki’s freeze-dried, Rikki’s non-freeze-dried), will be tested along with a sample from Rust Lab for comparison. *8 samples (2 from each group) retrieved from -80C freezer, thawing on ice *2uL DNA has been inserted into 4 tubes containing 60uL competent cells,, other tubes function as neg controls *Incubating on ice for 3 minutes, then heat shock for 1 minute. *Holding on ice for 5 more minutes *Incubating for 30min at 37C *Cells have been plated in CAM plates, incubating at 37C RK, KY *Made gel for PCR, two rows 14 wells *If we need to make gel use erlenmeyer flask labeled “Gel Flask” on the bench. *Updated protocols for making gel plates a bit. *PCR WORKED for most of the genes!!! I will update protocols since gDNA prep apparently works fine and one of the cycling conditions looks much better than the other. *Here’s a link for it: **https://www.dropbox.com/s/el29akgheqd7vre/UVP01768June202014.jpg RK *Made, filtered more than 1000 mL Tris HCl *Made, filtered more than 166mL EDTA *Created 1L Qiagen buffer P1(needs RNase) (Excess Tris HCL in 1L bottle, excess EDTA in 50mL tube) *Made, filtered more than 1000mL Qiagen buffer P2 *need to make N3 *(will add Qiagen buffers to protocol: http://openwetware.org/wiki/Qiagen_Buffers ) *Made gel plate for second gel electrophoresis *Performed restriction digest on plasmids RK, EJ *Looking up expected sizes for all genes to compare to PCR results. *2-mutY, 1053bp--did not work *3-ParC, 2259bp good *4-DinB, 1056bp good *5-MutS, 2562bp good *6-Dam, 837bp good *7-MutT,390bp good *8-Umu’C-1269bp good *9- EmrR-531 bp good *10- DnaE-3483 bp a little weird, some deposit around 550, 575 bp, should rerun (KY note: This is OK, its probably some other background amplification, we can get rid of it with fragment isolation) *11- MutL-1848 bp--did not work *12- MutH-690 bp--did not work *will ask Kevin for name of PCR software when he gets back *PCR worked much better on genes for the second condition (but harder to compare to ladder) *Try to avoid using Goldbio ladders: NEB ladders work better *For a 23X mastermix: **115uL 10X buffer **23uL dNTPs **23uL DNA stock **11.5uL Taq **954.5uL dH2O **1127 total -> 49 uL per tube, add 1 uL primers per tube **Tube numbers: 1: neg control, 2: ParC, 3: DinB, 4: MutS, 5: Dam, 6: MutT, 7: UmuC, 8:EmrR, 9: DnaE, 10: AroF, 11: Neg. control, 12: MutY, 13: MutL, 14: MutH (11-14: protocol 1) 15: Neg control, 16: MutY, 17: MutL, 18: MutH (15-18: protocol 2) 19: neg control, 20: MutY, 21: MutL, 22: MutH (19-22: protocol 3) **Protocol 1: extended 68 (2 minutes) **Protocol 2: 45 second instead of 30 second cycles **Protocol 3: 59 degree annealing temperature, extended 68 KY: *I will start the restriction digests on all the plasmids. We can fragment isolate everything on Monday and do all the cloning as well. There’s no reason it shouldn’t look good now that our PCRs turned out great (9/11). *I digested the following plasmids with enzymes S and P (will update master list later with protocol for restriction digests): PA, PC, 32A, 32C, 34A (all the minipreps from earlier) *I used the following protocol: **50 uL total volume **10 uL plasmid (except negative) **1 uL per enzyme (SpeI and PstI except no cut) **5 uL Buffer **33 uL H2O **Incubate for 1 hour at 37 degrees C; heat inactivate at 65 for 20 minutes; run a quick gel to verify the cuts (just **for this first one to make sure our enzymes are OK, I won’t do this in the future) **8X master mix: **40 uL Buffer **264 uL H20 **Total: 320 uL total -> 38 uL per tube, then add 10 uL plasmid *Gel lanes: 1- negative, 2- PA no cut, 3- PA cut, 4-PC cut, 5- 32A cut, 6-32C cut, 7- 34A cut *Add 1 uL per enzyme (Spel and PstI) (except for no cut) Things to do over the weekend: *Finish planning the timeline *Update protocols for gDNA, the multi-stage PCR, restriction digests, etc *Take out the PCRs from the machines *Check the transformants to make sure competent cells are OK (this and taking out the PCR should just take a few minutes at most) *Take out the MG1655 and delta-TyrR strains, put them in the fridge *(KY note to self: I need to pay that invoice tonight) Things to do on Monday: *Restriction digest, fragment isolate the genes *Restriction digest the linearized plasmids (pSB1C3, pSB1T3- do we have 2014 kit of these???) *Fragment isolate the plasmids *We need more miniprep columns. We’ll be doing at least 33 next week if everything goes well. *Glycerol-ize+freeze down the MG1655 and delta-TyrR so we have stocks of these strains in the future *We need Dpn1 and site-directed mutagenesis polymerase Things we borrowed: *Rust Lab: *8.09g NaOH *.01g SDS *6.06g Trizma *3.72g EDTA *1 1L vacuum filter *1 250mL vacuum filter *~5 uL SpeI *~5 uL PstI

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