Template:Waterloo/JS/notes

var notes = { "Sep13": [], "Sep12": ["Innoculate stuff for target characterization "], "Sep11": ["Transform batch 5 dcas9", "Run diagnostic gel of cpcr of dcas9 batch 4 "], "Sep10": ["Colony pcr the sgrna-gfp ngag, use 326 for positive ctrl, and don't forget negative control use vf2, vr (keep patch plates)", "Run diagnostic get fro sgrna-gfp ngag ", "Colony pcr the batch 4 of target biobrick, both t4 and tt4 using vf2 and vr primers (keep patch plates)", "Run diagnostic gel for batch 4 of target biobrick ", "Aliquate the dh5-alpha competent cells then flash freeze", "Transform new competent cells to test transformation efficiency ", "Start batch 5 of dcas9 take the fresh miniprep sample and dilute it so you use bw 1-10ng in pcr reaction amplify with 150,151", "Add 1ul of dpni and incubate at 37 for one hour. then column puirify", "Run a diagnostic gel for the dcas9 pcr", "Digest 181 with spei and psti and add 1ul of fastap (digest for 45 min)then column puirify (run 2-3 reactions and run them in one column)", "Digest the column puirified dcas9 with xbai and psti then column puirify", "Ligate digested dcas9 into 181", "Inoculating the dcas9 mutegenesis 2nd mutation round ", "Cpcr on batch 4 of dcas9 (pick 10 colonies) ", "Run diagnostic gel for dcas9 samples ", "Prepare samples for flow cytometry of target 1.0 biobrick ", "Run inoculated target on flow cytometry to characterize it with gfp and rfp ", "Make frozen stocks of target 1.0 biobrick ", "Miniprep target 1.0 biobrick and elute in water to send for sequencing (only use 3ml)", "Cut target 1.0 biobrick with x and p and digest for 45 minutes. cut psb1c3 as well for a control", "Run digest on a gel and also run uncut as well "], "Sep9": ["Miniprep the innoculated samples (total 10 tubes)", "Start batch 5 of dcas9 take the fresh miniprep sample and dilute it so you use bw 1-10ng in pcr reaction amplify with 150,151", "Add 1ul of dpni and incubate at 37 for one hour. then column puirify", "Run a diagnostic gel for the dcas9 pcr", "Digest 181 with spei and psti and add 1ul of fastap (digest for 45 min)then column puirify (run 2-3 reactions and run them in one column)", "Digest the column puirified dcas9 with xbai and psti then column puirify", "Ligate digested dcas9 into 181", "Transform all the ligations for ngag sgra gfp", "Transform ligations of target 1.0 batch 4 biobrick", "Transform ligations of bacth 4 dcas9", "Make competent dh5alpha cells and put in fridge ", "Design primers for ngag amplification", "Dcas9 mutagenesis on 4 good colonies from first round ", "Treat pcr products with 1ul of dpn1 for 1 hour", "Transform 10ul of pcr products and plate on lb amp plates ", "Inoculate target 1.0 biobrick for flow tomorrow and frozen stocks, also make streak plates "], "Sep8": ["Pcr dcas9 from the gel extracted pcr product starting batch 4", "Add 1ul of dpni into pcr product leave it at 37 incubator for one hour ", "Column puirify the dcas9 pcr product after being treated with dpni (each 3 rxn in one column)", "Run a diagnostic gel for the dcas9", "Digest 181 with spei and psti and add 1ul of fastap (digest for 45 min)then column puirify (run 2-3 reactions and run them in one column)", "Digest the column puirified dcas9 with xbai and psti then column puirify", "Ligate digested dcas9 into 181", "Digest target 1.0 with ecori and psti ", "Digest psb1c3 with ecori and psti ", "Run both of them on a gel for gel extraction", "Ligate the gel extracted target 1.0 into psb1c3", "Colony pcr of target 1.0 into biobrick", "Diagnostic gel for cpcr ", "Dcas9 mutegenesis pcr with 152, 153 on the already mutated. ", "Treat dcas9 products with 1ul of dpn1 for 1 hour", "Transform the mutated dcas9 into dh5-alpha using amp ", "Inoculate 111(cm), 181(cm), 322(km), 326(km), 335(amp) from frozen stock two tubes of each", "Miniprep 326", "Make 30 plates of cm", "Stuff tips", "Autoclave", "Make more lb agar", "Make 2 bottles of amp plates", "Inoculate dh5-alpha cells for making comp cells tomorrow", "Make solutions for comp cells tomorrow (0.1m cacl2, 0.1m mgcl2)", "Autoclave gsa bottles and prepare lb in large flasks for comp cells ", "Digest 1000ng 326 with spei and psti and fastap using purple fd buffer for 30min then column puirify (do 2 reactions and column puirify in one column)", "Digest gfp sgrna ngag using xbai and psti using fd buffer for 30 min then column puirify", "Ligate the gfp sgrna ngag into 326", "Order primers for sgrna flanking with prefix and suffix and the ones for ottawa"], "Sep4": ["Colony pcr of dcas9 batch3. have negative ctrl. use mini prep of 335 for positive. screen 5-10 colonies from one of the experimental plates", "Diagnostic gel of colony pcr ", "Colony pcr of gibson product with different primers ", "Diagnostic gel of colony pcr ", "Water plants"], "Sep3": ["Miniprep target 1.0", "Make frozen stock of target 1.0", "Miniprep dcas9 batch 3", "Make temporary frozen stock of inoculated dcas9 batch 3", "Diagnostic digest of cas9 batch 3. cut with x+p and use 181+335 as controls", "Diagnostic digest of target 1.0. cut with x+p and use 181+335 as controls", "Run diagnostic gel for above digests, include cut and uncut for all samples", "Water plants"], "Sep2": ["Send the envelope by getting it to lucy before noon. please take the taped tube out of the envelope. read the comment", "Take the transformation plates out of the incubator. can send the picture of the coloies to me. ", "If there are colonies do a cp pcr while keeping a patch plate. read the comment. cotact peivand for how may colonies to screen.", "Run 2ul of pcr product 3ul water 1ul dye on a gel for the colony pcr you should expect a band at kb you can send me the picture", "If the colony pcr did not work innoculate some colonies for more screening by digest by tomorrow", "Put all the minipreps from yesterday, label and date and say they are minipreps and store at ayan biobrick box. ", "Run the followig digested samples again on a gel. refer to the comment section. ", "Digest the following samples with x&p again leave it for 45min and run a diagnostic gel. label clearly and send the pic to peivand plz.", "From the batch 2 target biobrick streak bag take the streak plates pick a single colony and re-streak. ", "You probably will need to make more cm plates and maybe also make more lb agar?? i dunno", "Batch 3 cas9 innoculation"], "Sep1": ["Miniprep all the innoculations wash 3 times and elute in water these are for sequencing so they need to be extra clean. ", "Diagnostic digest of batch#1-dcas9-2 colonies 1-10 use x &p use fd green buffer", "Diagnostic digest of batch#1-dcas9-3 colonies 1-10 use x&p use fd green buffer", "Diagnostic digest of target#1 and target#2 batch2 colonies 1-5 for each use x&p use fd green buffer use 300-500ng", "Run as many gel needed to load all diagnostics from the digests above", "Transform batch#3 dcas9+181 plate on cm plates. ", "Prepare all samples for sequencing and submit the order through brian. hopefully all done before noon or before 3.", "Miniprep b1 & f2, elute in water w/ triple wash, nano for sequencing (2ml), use other 3ml for regular mini-prep.", "Gather all the reagents and make up stock solutions from them for western blotting.", "Gather all the equipment and troubleshoot (charles lab equipment is incomplete). ", "Prepare protoplasts for western blotting by concentrating them then boiling with 5x lysis buffer."], "Aug31": ["Miniprep the batch2 screens and nanodrop (x7)", "Run a diagnostic digest of sgrna_main + tar1.0 by cutting with x&p w/ tar1.0 as ctrl", "Run diagnostic gel of the digest screen, image", "Remove patch plate from oven, and put new tips/tubes in oven for drying", "Review oligo primers for antibiotic swap tests on benchling", "Literally re-do the negative control of the cpcr with exactly the same materials in the same spot.", "Run diagnostic gel of the -ve ctrl to check for contamination somewhere, image", "Run a flow on the sgrna_main_new (b1&f2) to check for good rfp & gfp expression", "Inoculate b1&f2 to make frozen stock of the new tar2.0.1", "Lab maintenance (ie. buy the brown napkin-towels, clean the sink and table)", "Make km200, cm200, km/cm; >10 each (plates)", "Send and confirm new primer order", "Amplify dcas9 3 tubes and run a diagnostic gel then column puirify everything into one column", "Cut the column puirified cas9 wirth x and p and column puirify", "Cut 181 with s and p and add sap then column purify", "Ligate dcas9 into 181 ", "Innoculate from streak plates for target1.0 into psb1c3 frozen stock", "Innoculate dcas9 for restriction digest test frozen stock", "Innoculate gfp-sgrna 1,2 ctrl+psb1c3 for se miniprep and frozen stock", "Innoculate rfp-sgrna #5+psb1c3 for frozen stock", "Water plants"], "Aug30": ["Order t4 ligase", "Colony pcr of dcas9 into 181 and make patch plate use 150, 151", "Run a diagnostic gel for cpcr of dcas9", "Streak target1.0+psb1c3 in cm ", "Cpcr of streak purified batch 2 product (ie. sgrna_main_new) w/ 128/129", "Diagnostic gel of cpcr product, image,patrick,done,success, question mark..", "Re-checked and designed primers for dcas9 mutegenesis, sgrna mainnew, antibiotic ressistence killers", "Inoculate agrobacterium from small inoculant into larger inoculation (250ml)", "Update the pcambia + pcocas9 + b1 + b2 + b3 sequence on benchling. ", "Design new sequencing primers for gibson pcambia"], "Aug28": ["Make 30 cm plates ", "Transform batch 1&2 of dcas9 into 181 and target1.0 in psb1c3"], "Aug27": ["Ligate dcas9 into 181 batch1 and target into 111 batch 1", "Transform batch3 main new into psb3k3", "Pcr dcas9 (dilute and vortex first) starting batch 2. use 4 reaction.", "Run a diagnostic gel and column purify the product", "Cut dcas9 with xbai and psti and column puirify", "Digest 181 with spei and psti and column puirify", "Run a diagnostic gel for both digests. ", "Ligate the dcas9 into 181", "Streak new main into target1.0 attempt 2.", "Digest target 1.0 with x and p", "Digest psb1c3 use x and p", "Run a gel and gel extract both ", "Ligate target 1.0 into psb1c3", "Make 2l of lb agar", "Make km stock", "Email cedarlane to follow up on the antibody", "Wash glassware", "Design primers for new main biobrick ", "Create new streak plates from the results of the agrobacterium electroporation to be turned into liquid innoculants tomorrow. ", "Do another round of electroporations on the electrocompetent agrobacterium with the gibson b1 and b3"], "Aug26": ["Send biobrick samples for sequencing", "Ligate dcas9 into 181 ", "Ligate digested target1.0 into psb1c3 old samples", "Transform the old samples for batch 2 rfp-sg gfp-sg and pco-cas9 biobrick ", "Dilute the primers received today", "Prepare mutated dcas9 samples for sequencing", "Writing up wiki", "Pcr-amplify more sgrna_main_new w/ 128&129 and -ve ctrl", "Cp, nano, and run diagnostic gel on pcr product.", "Cp, nano, and run diagnostic gel on pcr product.", "Digest sgrna_main_new w/ x&p, and tar1.0 (new) w/ s&p, incubate 45minutes", "Heat kill, column purify, and nanodrop", "Ligate sgrna_main_new w/ tar1.0"], "Aug25": ["Miniprep the fresh gibson b1 and b3 for agrobacterium electroporation", "Make frozen stock of gibson b1 and b3 from innoculate. ", "Make a 200ml bottle's worth of yep agar plates supplemented with rifampicin, gentamycin, and kanamycin. ", "If we can acquire mouse anti-flag antibody, new round of protoplasts transfection with cas9 and gibson b1/b3. wait for anti-cas9 othewise. ", "Aqcuire and inspect western blotting equipment from charles lab. ", "Make sure the buffers etc. for western blotting from moffatt are sufficient to run a western. ", "Electroporation of minipreped gibson b1 and b3 into electrocompetent agrobacteria. voltage of 1.25v.", "Created streak plate from gibson b1 and b3 innoculates for future use. ", "Miniprep 181 and 335", "Pcr amplify dcas9(335) use 4 tubes of 50ul reaction. (ask peivand before start) ", "Run a diagnostic and a gel extraction to gel extract dcas9", "Digest 181 using spei and psti and column puirify ", "Digest gel extracted dcas9 using xbai and psti and column puirify ", "Run a diagnostic gel for both digests", "Ligate dcas9 into 181", "Aliquate and flash freeze dh5-alpha comp cells", "Prepare rfp and gfp sgrna ctrl for sequencing by diluting and adding primers as well as sending the order to brian", "Get the biobar form signed by trevor and give the form to kathy", "Plant new seeds for protoplasts", "Prepare 30 cm plates", "Prepare 20 km200ul plates, and 10 km400ul plates.", "Remove tips from dry oven, and reorganize the tips in the cupboard", "Refill 70% ethanol spray bottle", "Miniprep tar1.0 extremely carefully, nano.", "Pcr-amplify more sgrna_main_new w/ 128&129 and -ve ctrl", "Cp, nano, and run diagnostic gel on pcr product.", "Diagnostic gel of cpd & cut tar1.0", "Digest sgrna_main_new w/ x&p, and tar1.0 (new) w/ s&p, incubate 45minutes", "Heat kill, column purify, and nanodrop", "Ligate sgrna_main_new w/ tar1.0", "Transform batch 1 into bl21(de3) w/ double -ve ctrl", "Analyze the data from the flow experiments and determine if iptg does actually induce rfp expression.", "Make frozen stock of tar 2.0.3 + back-up"], "Aug24": ["Fill all the pipette tips.", "Make dh5-alpha competent cells", "Buy ethanol, sphi, and ecori, small tips", "Design and order primers for dcas9 and antibiotic ressistance", "Make 15% glycerol calcium chloride", "Email brian the sequencing order", "Miniprep samples for sequencing: three washes, elute in water", "Emailed and followed up for primers. same with following up with pcr repair guy. ", "Check nano-drop values for the lost b2 before sending to ottawa (if it's any good). if not, lets order them a new one. ,erin/steven,done,nanodrop values were surprisingly good! sent to ottawa. if they, again, say the sample isn't amplifying like it should, we'll send them a new synthesized fragment. ", "Is something up with the sequencing facility? website down. results should be back: check up on it!", "Prepare protoplasts from yesterday for western - boil directly in sds.", "Autoclave protoplast media components - they contain sucrose/mannitol!", "Secure the components for running a western blot in the charles lab.", "Potential to use primary anti-flag antibody until our anit-cas9 arrives - look into it! ", "Inoculate gibson-b1 and gibson-b3 from patch plate.", "Innoculate 181 and 335 for dcas9 promoter swap", "Anneal together the gfp oligo primers w/ thermocycler", "Do a flow on the post-iptg (>12hr) batch (ie. tar1.0 & tar2.0.3-dcas9)", "Analyze the data from the flow experiments and determine if iptg does actually induce rfp expression.", "Resuspend the idt synthesized fragments in 50ul of te", "Pcr-amplfy the sgrna_main_new w/ -ctrl", "Run a diagnostic gel for the pcr amplification product, then cp & nano", "Run another diagnostic gel for the cpd products to compare.", "Digest tar 1.0 w/ s&p, & sgrna_main_new w/ x&p, all with sap", "Heat kill, column purify, and nano the digest products.", "Ligate the digest products w/ -/+ ctrl", "Make sob agar bottles (x5)", "Transform bl21(de3) w/ tar1.0 on iptg gradient w/ double -ctrl", "Inoculate the dcas9-tar2.0.3 into soc cm/amp/km w/ -ve ctrl", "Inoculate 322 into lb w/ km -ve ctrl", "Make sob triple selection + iptg plates (x10)"], "Aug23": ["Full protoplast run-though and imagining using the floursecent microscope for viability analysis. ", "Run pcr-amplified b2 on a gel (5ul of sample, 1ul of ld). if no band at ~600bp, run another pcr to amplify it b2 sgrna.,peter,done,peter, what did this look like in the end? ", "Re-transform the ligations of all the measurement parts: only transform 5ul this time and plate 100ul on one plate and the rest on another plate", "Talk to nathan or john about using the bohls lab plate reader for tomorrow", "Transform +ve and -ve controls from kit plates", "Innoculate dh5-alpha in all antibiotics and for tomorrow comp cells", "Autoclave media for comp cells", "Inoculate potential colonies for rfp,gfp sgrna biobrick for sequencing", "Do a flow on the induced bl21(de3) and expect a larger rfp signal.", "Induce the bl21(de3)-tar1.0 w/ iptg for 3hrs and shake at 200-250rpm", "Do a flow on the uninduced bl21(de3) and expect a gfp signal, no rfp.", "Anneal together the gfp oligo primers w/ thermocycler", "Prepare samples of mutated dcas9 for sequencing. ", "Design primers for antibiotic targeting w/ dcas9 for red track", "Make 2l of lb agar and 2l of liquid lb"], "Aug22": ["Transform 10ul of ligations of measurement parts into 50-100ul of dh5alpha and plate all cells on lb cm (6 tubes)", "Take out gibson plates from 37 incubator ", "Cpcr on gibson colonies using primers 148/149. melting temp of 68c and elongation time of 2:45. negative control and positive control using pco_cas9", "Run product on gel load 3ul product, 2ul h2o, 1ul ld.,patrick,pending,they're in the fridge, top shelf, pink rack -julia", "Inoculate dh5a into lb, no antibiotics, w/ -ve ctrl.", "Remove tips and tubes from the oven, and autoclave more 1.5ml mf tubes", "Lab maintenance (sweep floors, throw out recycling, clean sink, wipe down benches)", "Complete experimental design for swap destroyer track, including schedule of experiments.", "Inoculate tar2.0.3-dcas9 into sob w/ tsm (x3), w/ -ve ctrl"], "Aug21": ["Make calcium chloride and magnesium chloride for dh5-alpha competent cells. autoclave and put in the fridge", "Autoclave two 500ml baffled flasks and two gsa bottles", "Digest promoters with s and p and gfp with x and p then heat inactivate or column purify", "Ligate gfp and promoters"], "Aug20": ["Meeting with barb moffatt - acquired secondary anit-mouse antibody", "Order ethanol, and purchase baby stickers for mf tubes.", "Wash test tubes, autoclave.,peivand, patrick", "Prepare solutions for competent cells", "Streak dh5-alpha", "Dcas9 mutants: make frozen stocks of dcas9 inoculations de 1-6 (clones from d1135e) and rqtr 1-6 (clones from r1335q, t1337r)", "Dcas9 mutants: miniprep the remainder of the inoculations above for sequencing", "Miniprep 331, 2 tubes, can be combined", "Colony pcr batch 1 rfp, gfp, into psb1c3", "Run a diagnostic gel of the pcr", "Measurement: miniprep 101 106 117(promoters) and gfp", "Digest promoters with s and p and gfp with x and p then heat inactivate or column purify", "Ligate gfp and promoters", "Gibson assembly of pcambia and pco_cas9", "Treatment of gibson with dpn-1 for 1hr at 37c", "Transformation of the gibson product ", "Miniprep tar1.0 and nano (entire tube)", "Re-do miniprep of tar1.0 and nano (4ml)", "Co-transform tar 2.0.2 and dcas9 into bl21(de3) w/ -ctrl, on sob k/c/a", "Co-transform tar 2.0.3 and dcas9 into bl21(de3) w/ -ctrl, on sob k/c/a", "Flow cytometry w/ 284/326/322, save the results and use as a reference tool.,alicia, patrick"], "Aug19": ["Directing call with monsanto canada representative.", "Re-preparation of protoplast solutions based on dustin's recommendations.", "Run gel of yesterday's sgrna b3 diagnostic pcr. ", "Finalize primer design for cas9 -> pcambia after discussion with others about xbai-only ligation. ", "Amplify more b2. 66 degree melting temperature works for sure. q5 since this is going to ottawa.", "Diagnostic gel of b2 pcr for ottawa", "Pcr of pco_cas9 with gibson primers (148/149) use 0.5ul of dna (+1/10 dilution), 1.25ul of primers, 10ul of q5mm and 7ul dh2o (total=20)", "Diagnostic gel of pco_cas9 pcr on same gel as gel extraction", "Gel extraction of pco_cas9 and nanodrop", "Gibson assembly of pcambia and pco_cas9 (2 piece)", "Transformation of the gibson product ", "Design oligos for ngag mutagenesis sequences ", "Design sequencing primers for dcas9 mutants (2 primers) (is this the same task as above or are yours different oligos?.....)", "Mutagenesis: inoculate several colonies from transformation of dcas9 mutants (2 mutants) for sequenceing", "Xylose promoter: see if any of the cells on the streak plate glow green then inoculate 331 for miniprep", "Measurement: inoculate from transformation plates for miniprep", "Miniprep the already spined down psb1c3", "Transform batch#2 sgrna-rfp,gfp,main,cas9 into dh5-alpha", "Innoculate from streak plates for batch #1 biobricks sgrna-rfp,gfp ctrl", "Digest target 1.0 using x and p ", "Gel extract target 1.0 cuting the band at around 1kb", "Ligate gel extracted target 1.0 into already cut and gel extracted psb1c3", "Transform the dh5-alpha use 0.1ul target dna, 1ng target dna both into 50ul and 100ul of cells. also do negative control on all antibiotics cm,km,amp", "Finalize co-transformation protocol for swap destroyer", "Prepare 20% w/v glucose solution to make 500ml of soc", "Co-transform tar 2.0.2 and dcas9 into bl21(de3) w/ -ctrl, on sob k/c/a", "Co-transform tar 2.0.3 and dcas9 into bl21(de3) w/ -ctrl, on sob k/c/a", "Inoculate 284 (cm), 326 (km), and 322 (km)", "Transform tar 1.0 (322) into bl21(de3) and dh5a w/ -ctrl", "Perform flow cytometry on transformation efficiency product to test for gfp/rfp expression", "Order primers", "Arrange and hand in our pcr machine to get fixed"], "Aug18": ["Dry run - creation of protoplasts. if results look good, use miniprepped cas9 to perform peg-mediated transformation.", "Calculate transformation efficiency of the bl21(de3) ", "Co-transform tar 2.0.2 and dcas9 into bl21(de3) w/ -ctrl, on sob k/c/a", "Co-transform tar 2.0.3 and dcas9 into bl21(de3) w/ -ctrl, on sob k/c/a", "Check for whether to tranform onto sob or soc depending on what affects iptg induction.", "Order sgrna_main (new) from idt, as well as gfp destroyer primer pair for oligo", "Create 1l of sob media, autoclave at e6", "Create 500ml of tb, autoclave at e6", "Send pcr-prepped b2 samples to ottawa", "Send tar 1.0 and 2.0.1 for sequencing", "Transform to test dh5-alpha (0.1ng,1ng each into 100,50 ul) using target1.0 on km plate", "Perform site-directed mutagenesis on dcas9 w/ q5, following julia's protocol", "Treat pcr products with dpni for 1h at 37c", "Transform 10ul of reaction into 100ul of really awesome dh5alpha cells and plate all cells on lb amp plates", "Send gibson assembly results for sequencing. ", "Diagnostic pcr on the potentially successful gibson assemblies using primers to amplify b3.", "Run a gel of the diagnostic pcr of gibson asembly ^", "Xylose promoter stuff: make lb km plates with2% xylose and streak xylose promoter-gfp and positive control gfp (strains 326, 331, 332)", "Digest gel extracted cas9 and 3 sgrna using x and p and heat inactivate", "Ligate all the 4 constructs into gel extracted psb1c3", "Streak the rfp+gfp sgrna batch#1 colonies. ", "Spin down psb1c3"], "Aug17": ["Run gel of pcr amplified b2 to check its quality before sending to ottawa", "Prepare gibson assemblies 1 and 3 (the ones that looked good) for sequencing.", "Prepare the protoplast reagents in 50ml falcon tubes for tomorrow's protoplast experiments", "Miniprep enough pcocas9 to potentially use after tomorrow's protoplast work ", "Diagnostic gel for pcr products of 3sgrna with overhangs and pco-cas9", "Pcr amplify the sgrna main, rfp ctrl, gfp ctrl with overhangs and column puirify", "Pcr amlify pco-cas9 using 143, 145 primers", "Run a diagnostic gel for the pcr products", "Digest psb1c3 xbai and psti and sap", "Gel extract psb1c3 ", "After pcr diagnostic gel for pco-cas9 we either gel extract or column puirify", "Gel extract the 3 sgrnas", "Digest pco-cas9 and sgrna using x and p and heat kill and column puirify", "Ligate 3 sgrna swaps into psb1c3 ", "Ligate pco-cas9 into psb1c3", "Transform batch1 of sgrna into psb1c3 all 6 ligation + 1 negative ctl only cell for transformation+1 positive ctrl just target 1.0", "Inoculate 4 tubes of lb w/ 111 & cm", "Call preach and pay and get the refund", "Email back to the retailor for antibody quotation", "Send the po form for antibody to lucy", "Design sgrna for different pams", "Design primers for pco-cas9 classical cloning into pcambia+sgrna avoiding ecori", "Design sequencing primers of dcas9", "Order medium tips and sap and possibly bglii", "Ordered 400ul of gel red", "Update the finance spreadsheet", "Pcr amplify pcambia and pco_cas9 with gibson ends ", "Diagnostic gel for pcr today ", "Diagnostic gel from pcr from last week ", "Gel extract and column purify pcambia, pcambia-sgrna, and pco_cas9", "Make cm/ km plates for tranformation efficiency. (x24)", "Transform tar1.0 into bl21(de3) w/ +/- ctrl.", "Miniprep 322 and 338 inoculations and elute in water for sequencing", "Re-design gfp oligo pair w/ sphi restriction site.", "Re-design sgrna_main w/ sphi restriction site.", "Lab maintenance (ie. glassware, benchtops, garbage)"], "Aug14": ["Re-amplify b2 fragment to be sent to ottawa. ", "Innoculate gibson assemblies 1 and 3 for miniprep for sequencing for tomorrow.", "Innoculate pcocas9 for lots of minipreps for tomorrow. ", "Transform puc19 (0.1ng, 1.0ng) into 50ul x4 of bl21(de3)", "Combine target plasmid wiki w/ sgrna swap wiki.", "Inoculate tar1.0 and tar2.0.1", "Ligate psb1c3 with column puirified digests of sgrna rfp ctrl and gfp ctrl", "Pcr amplify sgrna3 rfp ctrl+gfp ctrl+main with overhang primers ", "Pcr cas9 using 143, 145 primers "], "Aug13": ["Miniprep innoculated psb1c3 (strain#111)", "Digest psb1c3 using xbai and psti", " gel extract the digested psb1c3 and extract for backbone", "Ligate psb1c3 with column puirified digests of sgrna rfp ctrl and gfp ctrl from last night"], "Aug12": ["Test digest of the miniprep samples cuting with xbai and psti ", "Diagnostic gel for the cas9+psb1c3 test digest (0.8%)", "Column puirify all the pcr products from last night, nano", "Run a pcr diagnostic gel by loading only 2ul of pcr product+3ul water+1ld", "Digest psb 1c3 with x & p then gel extract psb 1c3 ", "Digest the column puirified pcr products of the sgrna+overhang pcr using x&p then column puirify"], "Aug11": ["Miniprep the colonies of cas9+psb1c3 colonies #1,3,4,5,7", "Test digest of the miniprep samples cuting with xbai and psti ", "Diagnostic gel for the cas9+psb1c3 test digest (0.8%)", "Repeat pcr of sgrna main+control gfp+control rfp from synthetised part using 128, 129 primer and neb tm calculator using 2*taq. use 2 water control", "Pcr amplify sgrnas: 1.main(137,140) 2.gfp ctrl (138,140) 3. rp ctrl(139,140)", "Column puirify all the pcr products, nano", "Run a pcr diagnostic gel by loading only 2ul of pcr product+3ul water+1ld", "Run digest of of psb 1c3 with x & p then gel extract psb 1c3 ", "Pcr of pcambia (146/147), pcambia-sgrna (146/147) and pco_cas9 (148/149). use q5", "Run pcr samples from saturday on gel and gel extract pcambia, pcambia-sgrna, and pco_cas9", "Pcr diagnositc gel on pcr products from today ", "Grow dh5alpha to od 0.4, and bl21 to od 0.7,patrick,done,just chilling, holy crap dh5alpha is such a slow poke", "Centrifuge, wash, and resuspend in cacl2 o/n"], "Aug10": ["Pcr amplify sgrna main+control gfp+control rfp from synthetised part using 128, 129 primer and neb calculator for neb using taq", "Run diagnostic gel for the pcr product and column puirify rest of the reaction. ", "Repeat the diagnostic gel for the pcr product due to possible contamination in pre-cp diagnostic.", "Cp the pcr product of each tar2.0, nano.", "Inoculate from streak plates of pco-cas+psb1c3 colonies #3,5,1,4,7 in 5ml of lb+cm", "La1: miniprep target 2.0s, and nano.", "La1: digest each w/ x&p (200ng), incubate 45min", "La1: diagnostic gel to check if all the target 2.0s are really, really done, image", "La2: prepare co-transformation procedure for dcas9 and gfp_control, as well as rfp_control.", "Incolulate colony of bl21(de3) into soc liquid w/ cm", "Inoculate colony of dh5alpha into lb liquid.", "Remove bio-medical waste, and clean autoclave tray", "Prepare 0.1m mgcl2 (200ml) and 0.1m cacl2 (200ml), autoclave"], "Aug9": ["Put together the reagents in 1x stocks for protoplasts", "Transform 2ul of the gibson reaction into 100ul of dh5a - plate on kanamycin plates. ", "Diagnostic gel of the cas9 in the pcr of the gibson results", "Streak the lransformation product of pcocas9 and psb1c3", "Plan biobrick and divide work with erin and ayan", "La1: miniprep batch 3 trans-product, elute w/ 30ul eb, nano.", "La1: diagnostic digest using x & p, incubate for 45 minutes.", "La1: diagnostic gel of batch 3 digest", "La1: diagnostic pcr of sgrna_main w/ 128+129, tm=71, exttime=30s", "La1: diagnostic gel of batch 3 pcr", "La1: make frozen stock of batch 3 if the two tests are successful; self-destruct if failed.", "La1: inoculate 2.0.1, 2.0.2, 2.0.3 (338,339,340) into lb liquid w/ km", "La2: resuspend bl21(de3) in gly-cacl2 sln and flash freeze w/ n2", "La2: prepare co-transformation procedure for dcas9 and gfp_control, as well as rfp_control.", "La2: transform puc19 into cacl2 comp bl21(de3) to calculate transformation efficiency.", "Make 5 lb agar bottles", "Make 5 soc agar bottles", "Make 20 lb+cm plates", "Make 20 lb agar plates w/ km (200ul)", "Make 20 soc agar plates w/ km (200ul) + amp (100ul) + cm (100ul)", "Make 10 soc agar plates w/ cm", "Lab maintenance: clear fridge of all old antibiotic plates, wash media bottles for new agar", "Remove glassware from autoclave, and place in oven until dry.", "Streak purify dh5alpha stock from charles lab for comp cells (p49)"], "Aug8": ["La1: inoculate transformation product in lb liquid w/ km", "La2: make bl21(de3) comp cells and sit in ice and in fridge", "Make 15% cacl2 + 15% glycerol soln, and filter sterilize."], "Aug7": ["Pcr amplify pcambia and pco_cas9 using gibson primers (146/147 for pcambia, 148/149 for cas9)", "Run pcr products on a gel and if they look good cp and nanodrop", "Transform ligation product (cas9+psb1c3) w/ +/- ctrl", "La1: transform batch 3 w/+/- ctrl onto km ", "La2: make frozen stock of the bl21(de3)", "La2: prepare all solutions for comp cells tomorrow", "La2: inoculate 5ml soc w/ the bl21(de3), triplicate", "Label all tip boxes with igem and clean them out & reorganize them into proper sections in the cabinet."], "Aug6": ["Gel extract pco-cas9 pcr product from yesterday. load two lanes each contain 20ul of sample+4ul 6*dye", "Digest the gel extracted product using xbai and psti and heat inactivate pco-cas9", "Digest psb1c3 using xbai and psti ", "Gel extract digested psb1c3, nano", "Ligate digested pco-cas9 into sb1c3 use the ration of 3:1", "Miniprep pco-cas9, nano", "Pcr amplify pcambia and pco_cas9 using gibson primers (146/147 for pcambia, 148/149 for cas9)", "Diagnostic gel of pcr amplification of cas9 out of gibson colony product", "La1: pcr amplify stock of sgrna_main w/ q5", "La1: run diagnostic gel of sgrna_main pcr product.", "La1: cp and nano sgrna_main pcr product. self-destruct if row 23 is unsuccessful.", "La1: pcr amplify more sgrna_main from product of above amplification", "La1: run diagnostic gel of sgrna_main pcr product.", "La1: cp and nano sgrna_main pcr product. ", "La1: miniprep target 1.0 (2 tubes, all), nano", "La1: digest new sgrna_main w/ x & p, +sap, incubate for 45minutes, followed by pek", "La1: cp and nano digested insert.", "La1: digest newly minipreped tar1.0 w/ s & p, +sap, incubate for 45minutes, followed by pek", "La1: ligate tar 1.0 w/ sgrna_main", "La2: inoculate bl21(de3) into 5ml of soc each, duplicates, w/ cm"], "Aug5": ["Send samples for sequencing by noon.", "Innoculate pco-cas9 from frozen stock", "Pcr amplify pco-cas9 using taq and primer 143, 145", "Diagnostic gel of pcocas9 product, image", "Diagnostic pcr of gibson colony product, amplifying out sgrna.", "Diagnostic gel of pcr product above ^", "Diagnostic pcr of gibson colony product, amplifying out cas9.", "Diagnostic gel of pcr product above ^", "La1: run troubleshooting w/ pcr to test q5/128/129 contamination.", "La1: run diagnostic gel of troublshoot pcr products, image.", "La1: amplify stock of sgrna_main w/ taq hs", "La1: run diagnostic gel of amplification product, image.", "La2 streak bl21(de3) on two cm plates w/ - ctrl", "La1: inoculate 326 into two tubes w/ km", "Make new (2nd) red track box for new samples.", "Make 1l of sob media, and autoclave", "Make 50ml of 20% w/v glucose and autoclave", "Make 1l of soc media, making sure to filter sterilize the 20% glucose when adding to the sob media.", "Replace all milliq/ pcr milli q", "Re-arrange the 10um primer box in order"], "Aug4": ["Pcr amplify pco-cas9 using primers 143 & 144 run a diagnostic gel and column puirify use 1-10ng of dna using taq", "Diagnostic gel for today's pcr and diagnostic gel for yesterday again, run more sample", "Transfer the samples into plate or sequencing and send them before noon", "Finish lane 1 experimental plan for the rest of the week.", "Finish lane 2 experimental plan for this week and next.", "Wash test tubes and glassware", "Take the test tubes out of the autoclave and put them into the dryer", "Order reagents for protoplasts, 10ml pippette, q5 polymerase , lab book and ine tip sharpie from chemstore", "Talk to charles about western reagents", "Autoclave tubes, and well plates.", "Have 5 rules finished for the binding effects on aa stuff"], "Aug3": ["Miniprep and elute in water and prep the samples to be sent out for sequencing", "Pcr amplify pco-cas9 using primers 143 & 144 run a diagnostic gel and column puirify ", "Diagnostic gel for pcr", "Re-nanodrop cps from yesterday ", "Ligate pco_cas9 into both pcambia and pcambia-sgrna a2. no insert control and no ligase control ", "Miniprep samples for sequencing, remember to elute in water and wash 4 times "], "Jul31": ["Digest pcambia and pcambia-sgrna a2 with e&x and pco_cas9 with e&x, digest for 45 minutes", "Cp the digested products and then nanodrop ", "Inoculate pcambia samples for sequencing (total 6 samples)"], "Jul30": ["Re-transform gibson product w/ appropriate antibiotics", "Diagnostic gel of batch 1 digest using 1.5% gel for extra resolution at 80v.", "Diagnostic gel of batch 1 pcr using 1.5% gel for extra resolution at 80v.", "Diagnostic gel of batch 2 digest using 1.5% gel for extra resolution at 80v.", "Diagnostic gel of batch 2 pcr using 1.5% gel for extra resolution at 80v.", "Miniprep new dcas9 (strain 335) and elute w/ eb and nano.", "Make frozen stock of 335", "Make all primer stocks", "Wash all test tubes."], "Jul29": ["Ethanol precipitate and speedvac samples then nanodrop again", "Ligate pco_cas9 into both pcambia and pcambia-sgrna a2. no insert control and no ligase control ", "Miniprep 2ml of the batch 2 inoculations, and elute w/ 30ul of eb + nano", "Diagnostic digest of batch 2 w/ x&p (expect 2.7kb and 2kb band)", "Diagnostic gel of batch 2 digest using 1.5% gel for extra resolution at 80v.", "Diagnostic pcr of batch 2 to amplify out the rfp-gfp-sgrna_main", "Diagnostic gel of batch 2 pcr using 1.5% gel for extra resolution at 80v.", "Make frozen stock using the remaining 1.5ml of batch 2", "Inoculate bl21(de3) into lb liquid", "Streak more colonie target 1.0+ psb1c3 ", "Ensure we have access to all necessary enzymes and buffers for protoplast protocol."], "Jul23": ["Ethanol precipitate and speedvac samples then nanodrop again", "Ligate pco_cas9 into both pcambia and pcambia-sgrna a2. no insert control and no ligase control ", "Begin screening of transformation product batch 2 by inoculating into lb liquid w/ km", "Transform ligation product of sgrna_main in tar1 w/ +/- ctrl", "Miniprep 3ml of the batch 1 inoculations, and elute w/ 30ul of eb + nano", "Diagnostic digest of batch 1 w/ x&p (expect 2.7kb and 2kb band)", "Diagnostic gel of batch 1 digest using 1.5% gel for extra resolution at 80v.", "Diagnostic pcr of batch 1 to amplify out the rfp-gfp-sgrna_main", "Diagnostic gel of batch 1 pcr using 1.5% gel for extra resolution at 80v.", "Make frozen stock using the remaining 1.5ml of batch 1", "Inoculate bl21(de3) into lb liquid. no antibiotics."], "Jul28": ["Cp the pcr amplified swap destroyer parts and elute w/ eb + nano", "Streak purify new dcas9 onto amp plates.", "Run 2 diagnostic gel for both digest products and colony pcr products. or run a huge gel for both of them to visualise target 1.0", "Streak more colonies from target 1.0 ligation", "Miniprep entire volume of 181", "Prepare more lb agar bottles (x10)", "Prepare set of rules for structural and binding effects of amino acid changes (ie. bible)", "Prepare 10 plates w/ amp ", "Resuspend the new primers (targe1.0 sequencing)", "Lab maintenance (garbage, sink, floor),perin,done,sink done, floor done, garbage done,", "Prepared 500ml of 70% ethanol for spray bottle.", "Prepare samples for sequencing", "Finish half of wiki for red track", "Digest 3000ng pcambia with ecori using sap", "Calculate sample combinations for equimolar amounts for gibson of b1 b2 b3 pcocas9 and pcambia", "Speedvac and nanodrop all samples for gibson (including b1+b2 and b2+b3 pcr oe products) from calculations above", "Gibson reactions themselves (b1 + (b2+b3) + pcocas9 + pcambia), (b3 + (b1+b2) + pcocas9 + pcambia), and (b1+b2+b3+pcocas9+pcambia)"], "Jul27": ["Miniprep pcambia and pcambia-sgrna a2. total of 6 tubes ", "Nanodrop the 6 tubes ", "Digest pcambia and pcambia-sgrna a2 with e&x and pco_cas9 with e&x, digest for 45 minutes", "Cp the digested products and then nanodrop ", "Ligate pco_cas9 into both pcambia and pcambia-sgrna a2. no insert control and no ligase control ", "Transform ligation product of sgrna_main in tar1 w/ +/- ctrl", "Begin screening of transformation product batch 1 by inoculating into lb liquid w/ km", "Inoculate lb liquid with streak purified bl21(de3)", "Double digest: target 1.0 (322) w/ x&p, sgrna_main w/ s&p (duplicate)", "Cp: digest product of target 1.0 and sgrna_main", "Ligate: cpd target 1.0 (vector) with cpd sgrna_main (insert) **+/- ctrl", "Prepare samples for sequencing", "Fill up more small tips and run autoclave asap", "Finish half of wiki for red track", "Make 20 km plates (200ul) and 10 for (400ul)", "Classify amino acids into their primary and secondary subgroups, as well as rank them on their size.", "Mini prep 285", "Pcr of 285", "Inoculate 181 again", "Miniprep the 6 colonies of target1.0+ psb1c3 (note the two samples that are placed seperately, those are sketchy their caps fall off)", "Nanodrop the 6 tubes ", "Diagnostic digest of the minipreps of target1.0+psb1c3 using x and p only 100-300ng of digest is enough", "Run a diagnostic gel to visualise the insert. ", "Colony pcr of target 1.0 into psb1c3 streak plates (total of 6 colonies) use primers 24 and 25 (from last year, vf2 and vr) for the pcr and neb calculator", "Run the pcr product on gel and look for about 1 kb band", "Column purification of b2 pcr products for pcr oe. ", "Pcr oe of b1+b2 and b2+b3 using newly amplified b2. "], "Jul26": ["Inoculate pcambia (10ul km) and pcambia+sgrna (10ul km) into 5ml of lb for miniprep tomorrow. take 3 colonies from each plate ", "Design primers for gibson amplification of pcambia and pco_cas9 (set 2)", "Send snapgene files to uottawa", "Double digest: target 1.0 (322) w/ x&p, sgrna_main w/ s&p (duplicate)", "Cp: digest product of target 1.0 and sgrna_main", "Ligate: cpd target 1.0 (vector) with cpd sgrna_main (insert) **+/- ctrl", "Remove transformation plates from inciubator and streak purify.", "Streak purify bl21 into lb w./ no antibiotics.", "Design mutagenesis primers for new dcas9", "1. run gel of new pcr oe (b1 + b2 and b2 + b3) gel extracted products along side the new pcr of gibson cas9. ", "2. if gel in #1 looks good, digest 2000ng of pcambia with ecor1. use sap in the reaction.", "3. gibson assembly of pcambia, gibson pcocas9, b1, b2, and b3 (finally). ", "Pcr amplify more b2 for tomorrow morning's pcr oe reaction. ", "Colony pcr of target 1.0 into psb1c3 streak plates (total of 6 colonies) use primers 24 and 25 (from last year, vf2 and vr) for the pcr and neb calculator", "Run the pcr product on gel and look for about 1 kb band", "Innoculate the 6 colonies of target 1.0 into psb1c3", "Inoculate 285 (dcas9-278 in psb1c3) and 181 into 7ml lb cm for miniprep", "Order the primers", "Send samples for sequencing. target1.0, target rfp control, target gfp control", "Make more km antibiotic stock, remember to filter sterilize it", "Make more lb blank plates (no anti)", "Makre more 200ul km plates", "Make more cm plates", "Make more 400ul km plates (1 bottle worth)"], "Jul25": ["Convert dna sequence of b1,2,3, pco_cas9, pcambia 1305.1 to a snapgene file for uottawa", "Streak strain 312 and 316 from frozen stock "], "Jul24": [], "Jul23": ["Convert dna sequence of b1,2,3, pco_cas9, pcambia 1305.1 to a snapgene file for uottawa", "Run a gel of the pcr products from yesterday's re-amplification. if quality is good enough, gel extract from pcr oe products. ", "Design primers for gibson amplification of pcambia and pco_cas9", "Cp: pcr amplification product + nano", "Transform ligation product (ie. target 2.0.1) incubate for 1hr if time permits. **+/- ctrl", "Streak bcl21 from frozen stock.", "Write-up shad valley protocol for documentation.", "Design site-directed mutagenesis primers for s.py(?) dcas9", "Mini prep 285 and 181", "Pcr of 285 and the promoter+rbs from 181", "Digest 322 target 1.0 use xbai and psti and gel extract", "Ligate gel extracted 322 insert into psb1c3 vector+", "Organize the project track boxes."], "Jul22": ["Run a diagnostic gel on re digests. make sure to load the uncut plasmid from the minipreps as well ", "Design primers for gibson amplification of pcambia and pco_cas9", "Send minipreps from yesterday for sequencing (#1-3)", "Convert dna sequence of b1,2,3, pco_cas9, pcambia 1305.1 to a snapgene file for uottawa", "Pcr amplification: sgrna_main, sgrna_gfp-ctrl, sgrna_rfp-ctrl w/ primers 128/129 & q5", "Gel: diagnostic of pcr amplification product. [1ul of product+1ul 6x ld+4ul dh2o]", "Cp: pcr amplification product + nano", "Double digest: target 1.0 (322) w/ x&p, sgrna_main w/ s&p", "Cp: digest product of target 1.0 and sgrna_main", "Ligate: cpd target 1.0 (vector) with cpd sgrna_main (insert) **+/- ctrl", "Transform ligation product (ie. target 2.0.1) incubate for 1hr if time permits. **+/- ctrl", "Streak bcl21 from frozen stock.", "Aliquot cell lysis solution into six 1.5ml mf tubes.", "Load metal cart with: brian (gel dock), gel tray, gel lid, pre-made 1.1% agarose gel, five 100/200ul pipettes, five medium sized tip boxes, five waste beakers (labelled).", "Remove parafilm seal from transformation plates.", "Miniprep 322 target 1.0", "Digest 322 target 1.0 use xbai and psti and gel extract", "Ligate gel extracted 322 insert into psb1c3 vector", "Design primers", "Design site-directed mutagenesis primers for s.py(?) dcas9", "Mini prep 285 and 181", "Pcr of 285 and the promoter+rbs from 181", "The gibson reaction preparation:", "Nanodrop all the potential reaction components for gibson assemblies. ", "As a result of bad quality components: pcr of b2, pcr of pcocas9 (gibson primers), and pcr oe of b1+b2 and b2+b3. ", "Begin to develop the ultimate spreadsheet with a calculator for all recipes for all basic protocols", "Organize the project track boxes.", "Remove tips and test tubes from oven racks in the autoclave room, and charle's lab.", "Make 10 lb liquid and 10 lb agar bottles.", "Make more 50x tae (1000ml would be nice)", "Make 500ml of binding buffer"], "Jul21": ["Miniprep inoculations in the 37c incubator (pcambia+cas9+sgrna). wash minipreps #1-3 4 times and elute in water so we can send for sequencing", "Nanodrop of the 18 minipreps.", "Digest 200ng of each with e&x, and 600ng with x&p ", "Run a diagnostic gel on re digests. make sure to load the uncut plasmid from the minipreps as well ", "Design primers for gibson amplification of pcambia and pco_cas9", "Transform main destroyer ligation product into dh5alpha.", "Make frozen stock of target 2.0_rfp and target 2.0_gfp", "Streak bcl21 from frozen stock.", "Transform target 1.0 into dh5alpha w/ +/- ctrl (x5)", "Digest psb1c3 w/ x&p and cut 322 w/ x&p, run diagnostic gel", "Gel extracted psb1c3 and target 1.0", "Ligate 322 insert into psb1c3 vector", "Design primers", "Innoculate 322 target 1.0 km resistence", "Inoculate 285 (dcas9-278 in psb1c3) and 181 into 5ml lb cm for miniprep", "Design site-directed mutagenesis primers for s.py(?) dcas9", "Organize samples in freezer into their respective track box. ", "Begin to develop the ultimate spreadsheet with a calculator for all recipes for all basic protocols", "Clean test tubes and autoclave.", "Pcr overlap extension of b1+b2 and b2+b3 with modified melting temperatures"], "Jul20": ["Cpcr on the streak plates of pcambia-cas9-sgrna(a2) from yesterday, in 37c incubator. use primers 124/125 and anneal 68c (temp gradient), elongation 2:45", "Inoculate the 20 streak plates. (do not inoculate #18,19)", "Run a gel on cpcr products to check for insert ", "Digest main destroyer with x&p and target plasmid (322) with s&p, digest for 30 minutes ", "Cp the digested products and nano", "Ligate digested main destroyer with digested 322 ", "Inoculate patches from successful ligations of rfp_control and gfp_control so we can make a frozen stock tomorrow", "Digest psb1c3 w/ x&p and cut 322 w/ x&p", "Ligate 322 insert into psb1c3 vector", "Pcr oe of b1 + b2 and b2 + b3 followed by column purification at the 1200bp mark. ", "Gibson assembly attempt using the b-set. look in comments -->", "Update advanced protocols w/ low concentration ligation protocol, and cpcr protocol."], "Jul18": ["Pcr of b1, b2, and b3 from the original stock (to replenish). use tm = 64 and extension time of at least 20 seconds. 0.5ul of template should be enough.", "Run diagnostic gel to check badn size of pcr amplification of b1,2,3", "Run diagnostic digest test of target 2.0_batch1 (a5, b5, c1) w/ x&p & incubate for 40 minutes.", "Run diagnostic gel of double-cut target 2.0_batch1", "Run diagnostic digest test of target 2.0_batch 2 (a5, b5, c1) w/ x&p & incubate for 40 minutes.", "Run diagnostic gel of double-cut target 2.0_batch 2", "Miniprep the inoculated 183 ", "Inoculate strain 185 (-86 freezer in 377a, box 3)", "Make >21 lb plates with km ", "Streak 20 plates from the plate with the transformation product on km (200ul)", "Transform the pco_cas9 &pcam/pcam+sgrna_a2", "Digest 284 w/ x&p and 326 w/ s&p (1000ng of dna)", "Gel extract 1kb band from 284 digest, w/ cp & nano", "Cp the 326 digest product and nano. split them equally into 6 tubes.", "Save 10 tubes for dh5alpha for shad", "Update advanced protocols w/ low concentration ligation protocol, and cpcr protocol."], "Jul17": ["Miniprep the inoculated colonies of target 2.0 and nano "], "Jul16": ["Miniprep the inoculated colonies of target 2.0 and nanodrop (expect red in test tubes).", "Inoculate the patch-plated colonies (a5, b5, c1) into liquid lb w/ km & ctrl,patrick,done,6 tubes in total, this is batch 2", "Run diagnostic digest test of target 2.0_batch1 (a5, b5, c1) w/ x&p & incubate for 40 minutes.", "Run diagnostic gel of double-cut target 2.0_batch1", "Miniprep 111, no patch plate.", "Diagnostic digest + gel - cut 300ng of the miniprepped 111 in the following ways: ecori+psti, and xbai+spei. use 0.3ul of each enzyme. load 100ng into gel", "Streak plated psb1a3 (strain 183) onto two amp plates.", "Cpcr the transformation of cas9 into pcam/pcam+sgrna_a2 (make patch plates)", "If cpcr is good, inoculate reamining colonies in transformation plates into liquid lb w/ 5ul of km each.", "Please re-label the pcam/pcam+sgrna_a2 tubes.", "Digest pco_cas9 & pcam & pcam+sgrna_a2 with e & x, followed by cp", "Ligate pco_cas9 into both versions of pcambia ", "Make a dh5alpha box in the -86 freezer for the igem tower.", "Cpcr the dcas9_278 transformation product. ", "Run cpcr on gel", "Order the necessary primers for sequencing of target2.0.", "Update advanced protocols w/ low concentration ligation protocol, and cpcr protocol.", "Finalize protocol to create protoplasts because the sgrna swap destroyer experiments are almost ready to begin.", "Begin to develop the ultimate spreadsheet with a calculator for all recipes for all basic protocols", "Stuff 200ul (medium) tips and autoclave plz."], "Jul15": ["Miniprep 111 (we have to miniprep on friday because we didn't get a lot of growth from the frozen stock) ", "Perform colony pcr screening tests on the newly transformed colonies with target 2.0. (make patches)", "Run diagnostic gel for the colony pcr products.", "Gel extraction of b1+b2 and b2+b3 samples. extract between 1.0kb and 1.4kb and load extra ladder (10ul). don't forget a pinch of guanosine. nanodrop.", "Pcr amplify more b1, b2, and b3. use 0.5ul of template, melting temperature of 64, and extension time of 20 seconds. ", "Run diagnostic gel, in parallel with gel extraction, of the pcr amplified b1,2,3.", "Diagnostic digest + gel - cut 300ng of the miniprepped 111 in the following ways: ecori+psti, and xbai+spei. use 0.3ul of each enzyme. load 100ng into gel. ", "Inoculate psb1a3 in liquid media so we can make a frozen stock (plates are in the fridge, try to do this in the afternoon)", "Patch colonies from transformation of dcas9-278 ligation", "Run a gel of new pcr amplified a1 (from erin's pcr yesterday)", "Inoculate the 111 plate into lb "], "Jul14": ["Measure transformation efficiency of comp cells ", "Transform the ligated product into dh5alpha w/ +/- ctrl for transformation", "Design primers for target 1.0 sequencing.", "Transform dcas9-278 ligations into dh5alpha?", "Digest pco_cas9, pcambia sgrna-a2 and pcambia with e & x ", "Ligate pco_cas9 into both versions of pcambia ", "Inoculate 111 in liquid media ", "Design and order primer to amplify sgrna a1 (backup)", "Run a diagnostic gel of yesterday's pcr amplification of sgrna a1 and a2, then column purify and nondrop each", "Pcr overlap extension between a1 and a2 after column purification", "Check quality of gel-extracted, pcr-cleanup b1+b2 and b2+b3. pcr oe between them and their third sgrna piece. ", "Send parts and primers to ottawa and a bunch of stuff for sequencing"], "Jul13": ["Get n2 from chemstore and aliquot competent cells into 400ul portions and flash freeze", "Transform puc57 into the dh5alpha cells and incubate o/n for transformation efficiency", "Digest dcas9-278 with x&p, digest 181 w/ x&p, as well as s&p, followed by hi", "Ligate o/n dcas9-278 into 181 cut w/ x&p (makes dcas9 cds biobrick) and s&p (makes dcas9 expression cassette biobrick)", "Make new dilution of 124, 125 primers to 10ng/ul", "Pcr amplify the pco_cas9 out of their backbones with 124, 125. ", "Diagnostic gel for the pcr of the fresh pco-cas9 and if it looked good column puirify or gel extract", "Digest 322 w/ s&p, and digest amplified sgrna_gfp/rfp controls with x&p", "Cp the digested products. ", "Ligate the inserts into the backbones (ie. gfp control into 322, rfp control into 322) w/ +/- ctrl", "Design primers to amplify pco_dcas9 in order to make biobrick (put it into psb1c3)", "Re-do a1, a2 amplifications.", "Cp a3 and a4 since they looked good (although late)", "Gel extraction and pcr cleanup of the b1+b2 and b2+b3 stuff."], "Jul12": [" miniprep 322", "Pcr amplify sgrna swap materials using primers 128/129 ", "Run products on a gel to make sure they are correct size ", "Cp and nanodrop products to keep", "Miniprep pco_cas9.", "Make the dh5alpha comp cells.", "Miniprep transformation screening cas9 pcambia ", "Digest with e&x for cas9 appearance, digest with x&p for sgrna-a2", "Digest pcambia+sgrna-a2 with e&x, and pcambia alone digest with e&x then column puirify", "Run a bunch of samples on a gel (pcr oe samples)"], "Jul11": ["Inoculate dh5alpha competent cells into lb ", "Make frozen stock of psb1a3", "Miniprep the pco_cas9 e.coli from the liquid media ", "Inoculate the pco_cas9 e.coli that have been streak plated into liquid media.", "Inoculate 322 into lb liquid media", "Inoculate yesterday's streak plates of cas9 transformation into liquid lb", "Streak strain 111 (from frozen stock)"], "Jul10": ["Make 15% glycerol solution with 0.1m cacl2", "Streak plate dh5aplpha.", "Ran diagnosic for pcr clean up of cas9", "Ran diagnostic for pcr product of amplified dcas9-278", "Inoculate pco_cas9 e.coli into 10 tubes of lb liquid media with amp.", "Steak plate pco_cas9 e.coli onto 2 plates of lb with amp", "Streak plate with target plasmid (strain 322)", "Run steven's gel"], "Jul9": ["Make more lb agar (10 bottles = 2l)", "Miniprep 278 and 181", "Pcr amplify dcas9 out of the backbone from 278 ", "Pcr amplify cas9 out of the backbone from pco_cas9 minipreped sample.", "Run diagnostic gel of pcr product of cas9 and dcas9-278", "Make 15% glucose solution"], "Jul8": ["Pcr amplify more cas9re w/ 124 & 125", "Parallel diagnostic/extraction gel for cas9re product", "Digest with e&x along with the pcambia/pcambia-sgrna_a2", "Ligate the digestion product.", "Streak purify the tranformation product of cas9 into pcambs if successful.", "Inoculate one colony from the streak plate of psb1a3 (in incubator)", "Sgrna experimental design!!", "Make km antibiotic stock, followed by 10 of 200ul km plates & 10 of 400ul km plates.", "Make more lb agar (10 more bottles).", "Pcr amplify the swap destroyers (main + gfp/rfp controls) with primers 128 and 129", "Oepcr of b1,2,3 followed by amplification.", "Wash test tubes!!", "Prepare sgrna b1, 2, 3, gibson-amplified-pcocas9, and a copy of pcambia to be sent to ottawa"], "Jul7": ["Wash test tubes", "Streak puirify the psb1a3 on amp plate", "Pcr cas9re using 124 and 125 using 10ng and use q5 pcr master mix", "Run a diagnostic gel for the pcr product", "Run a gel for gel extracting cas9re ", "Cut today's attempt of cas9re gel extracted to cut with ecori and xbai using tango heat inactivate using patrick's heat kill program", "Ligate the product above using patrick's recipe", "Transform yesterday's ligation of pcr cas9re use all controls. for positive control use pcambia", "Pcr amplify b3 with correct primers..", "Make new lb agar", "Make new plates (km and amp)"], "Jul6": ["Remove 10ul tip boxes and mf tubes from oven in trevor's lab.", "Digest cas9-re, pcambia-sgrna_a2 and pcambia with correct res.", "Ligate cas9-re into pcambia-sgrna_a2 and pcambia using black magic ligation recipe.", "Transform ligation products in dh5-alpha", "Heat-inactivate some of the ligation product using p.e.k. thermocycler program.", "Digest the ligation product using single-cutter re and run a diagnostic gel.", "Run diagnostic gel on b1, b2, b3 amplifcation from yesterday, in parallel with an extraction gel to remove sgrna 650bp fragments.", "If b3 pcr was successful, pcr overlap extention between pcr amplified b1, b2, and b3.", "Begin attempt 3 of construct attempt by pcr amplifying more cas9 with re sites.", "Gel extract the 5kb fragment, followed by cp+nano", "Annotate rfp-gfp target plasmid on benchling", "Design primers around dcas9 from strain #278 flanking with xbai and psti as an attempt to improve on last year's biobrick", "Finalize experimental procedure for testing the swap destroyer.", "Transform psb1a3 into dh5alpha use both ligation reactions done yesterday a and c and their controls+positive and negative ctrl", "Make more km antibiotic stock, as well as 200/400ul km plates.", "Pcr amplify pcocas9 with gibson primers (108+109)", "Run diagnostic gel for pcocas9-gibson", "Look into making home-made miniprep solutions.", "Lab maintenance (someone needs to wipe down these dutty lab benches and re-organize the tips-stick cupboard.)"], "Jul3": ["Stuff 10ul pipete tips and fill new jar with 1.5ul microfuge tubes. autoclave asap. ", "The minipreped and digested psb1a3 that sam and erin did looked good. so we can transform that on monday.", "A little on my nerve when the registry protocol does not work i think it is from the wrong digest buffer. so let's cut psb1a3 with e/p using tango and column puirify. ", "Ligate the above (self ligating psb1a3) need a control of no ligase.", "Repeat the psb1a3 digest and ligation, but this time with the new emm (tango)", "Repeat the cas9new re cloning by pcr pco-cas9 using 10ng of dna in 50ul reaction and using primers 124 and 125 (dilute)", "Run diagnostic by loading 1.5 ul of pcr product and run the rest of the pcr product (close to all of it) for gel extraction", "Cut the cas9newre using ecori and xbai and use heat inactivation and tango buffer", "Ligate the above by adding atp and ligase (ask the specifics from patrick) ", "Retype cherry's protocols for agrobacterium in advanced protocol google doc", "Retype and update antibiotic list. (ask peivand about new updates) ", "Let's think of lab consumables needed for next two weeks and purchase them tomorrow. so we can stock up for the time that i am away on conference", "Thaw and filter sterilize our amp and km antibiotics and if needed make more stocks. ", "Design primers around dcas9 from strain #278 flanking with xbai and psti as an attempt to improve on last year's biobrick", "1. run diagnostic gel of pcr reactions from yesterday (sgrna b3 + gibson-pcocas9). load 4ul of sgrna b3 pcr product and 1ul of gibson-pcocas9. samples are sitting in fridge on pink pcr-tube tray.", "Pcr amplification of the already-amplified sgrna b1 and sgrna b2. we want even more copies of these guys to send to u-ottawa. ", "Re-run pcr amplification of sgrna-b3 with primers 122 & 127"], "Jul2": ["Transform the ligation from psb1a3 (both ligation and ligation control) also do transformation negative control (only cells) and use transformation positive control using strain #33 in peivand and akshay box. the concentration of plasmid 33 is 207.7 and use 50 ng. call peivand if you can't find the tube or if you have any question. there should be a total of 4 plates for your transformation.", "Miniprep psb1a3 innoculated last night by ayan tube is on 37 shaker", "Test digest the miniprep results by cutting it with ecori and psti (two single digest and one double digest)", "Lauren from the tube that you made frozen stock of the target did you miniprep any? if yes did you run a test digest?", "If we did not do a test digest of the miniprep target and we have the miniprep let's do a test digest by cutting it with xbai and psti check the lab book for confirmation"], "Jun29": ["Due to my nature of experiments today for brian i will not be in the igem lab today. i'll be at mc but you can reach me by text.", "Wierdly enough the transformation for psb1a3 did not work maybe a plate was messed up so needs ro be repeated. follow the instructions on the link", "Help steven with his pcr amplifications if needed", "Innoculate from psb1a3 frozen stock"], "Jun29": ["Repeat the test diagnostic digest from june21st (pat's) to check if sgrna_a2 is inside samples (7 digests w/ x&p)", "Test digest the circles for cas9 by cutting with e&x (2 digests)", "Test digest the squares for cas9 by cutting with e&x (7 digests)", "Run diagnostic gel on the test digests.", "Run diagnostic gel on the test pcr", "Diagnotic pcr using cas9 primers (124,125) w/ +/- control.", "Transform the self-ligation of psb1a3", "Make frozen stock of dh5alpha containing target. ", "Miniprep more of the target plasmid and run diagnotic digest/gel.", "Autoclave test tubes.", "Make rif and gm antibiotic"], "Jun28": ["Miniprep party of all the colonies from the cas9 into pcambia and cas9 into pcambia+sgrna-a2 results (about 10 maybe more)", "Test digest of miniprep results cut each cas9+pcam+sgrna-a2 by ecori and xbai, also cut with xbai and psti", "Test digest of miniprep results cut each cas9+pcam with ecori and xbai ", "Run a diagnostic gel for you test digest, make sure you are loading your uncut. ", "Make 300ml of 50% sucrosesolution and filter sterilize", "Keep checking the agrobacterium od & remind peivand to get silwet from dr. moffat", "Start the floral dip as soon as agro reached the od (i'm expecting around 3) ", "Innoculate from the streak plate of the target for frozen stolk (yeap let's do it the proper microbio way) we test one tube tomorrow and make frozen stok of the rest", "For psb1a3 backbone we need to resuspend i guess then digest use the lin to the protocol and ligate", "Innoculate colonies that looked good for cas9 after today's test from the streak plates. (add 5ul km into 5ml) "], "Jun27": ["Digest around 500-600 ng of agrobacterium with pcambia and pcambia+sgrna-a2 in them", "Run the digest again on the gel with a lot more dna so we can visualize the insert since we were not able to on saturday", "In parallel maybe miniprep the rest of the clture of agro (mae sure you wash 4 times)", "Innoculate the streak plates for cas9 into pcambia and cas9 into pcambia+sgrna-a2 ", "Innoculate target construct from the target plate so we can make frozen stocks tomorrow", "Let's figure out how to resuspend dna from registery plates and use the lin for protocol to digest psb1a3 and ligate so we can transform tomorrow", "Subculture agrobacterium into 250ml flasks (needs to be done around 4 pm) and i think 1:200 dilution should be safe"], "Jun26": ["Miniprep 2ml of the 6 samples of agrobacterium and wash 3 times", "Digest the samples minipreped and run it on a gel", "Innoculate samples in 2ml in 3 different dilutions and store at 28 degrees"], "Jun25": ["Cp + nano + diagnostic of pcr product hiding in patrick's pcr box (1,2,control in ricardo),peter,done,whats cp? column purify gracias", "Design new tests to check if the previous pcamb+cas9 transformation attempt #2 was successful.", "Run diagnostic digests for pcamb+cas9 tranformation attempt #1", "Run diagnostic gel for the pcamb+cas9 digests.", "Re-inoculate gfr/rfp target and make frozen stock.", "Re-inoculate psb1a3 from stock.", "Design better system for storing samples currently in use. ", "Do an inventory check on all enzymes and buffers that we have in fiona (all)", "Re-distribute the t4 dna ligase buffer into small volumes (50ul) to prevent atp degradation", "Transform the ligation reaction of cas9newre into pcambia. make sure to use both positive and negative control. total of 10 plates.", "Make 1l of yep media in the baffled flasks for the dip+1l of yep agar+1l of lb agar", "Make 300 ml of 50% sucrose+ 300 ml of 100mm cacl2", "Autoclave the baffled flasks+big centrifuge bottles", "Peivand needs to go to moffat's lab and design pcambia primers", "Calculate the transformation efficiency based on the transformation from yesterday ", "Innoculate agrobacterium for the dip"], "Jun24": ["1) miniprep the samples a, b, c,d for the pcambia+ sgrna-a2 ", "2) digest the gel extracted cas9 using ecori and xbai (30-35 min digest is ok)", "3) cut regular pcambia with xbai and ecori also cut pcambia+sgrna-a2 with ecori and xbai as well", "4) ligate the cut cas9 into both pcambia and pcambia+sgrna-a2 (show me the ligation reaction, let's see if we can use 4:1 or 5:1 ratio) ", "5) pcr amplify oepcr product from last night, followed by diagnostic gel.", "6) do transformation efficiency by transforming in dilutions using 284 and cm plates. (since we already have those plates", "7) dilute and get the part psb1a3 from the registry and transform today so it can be tested tomorrow and innoculated and make frozen stolk of it."], "Jun23": ["1) control didn't look good so repeat the pcr of cas9 using primers 124, 125. using two buffers for phusion and maybe the master mix? annealingtemp seems to be best around 61.3. negative control for all individual buffer is necessary", "2) run a diagnostic of pcr reaction load 1-1.5 ul if looked good either gel extract or column purify", "3) digest the product above and pcambia, column purify and ligate if the results of above sound promising", "4) electroporate pcambia alone and pcambia+sgrna a2 into agrobacterium+negative control", "5) check the dh5-alpha plates, count colonies and calculate transformation efficiency", "1. gel extraction of sgrna 123/234 (ask patrick why it's called this and where to get it :p), followed by pcr oe with sgrna 1 and 4 (use c1 and c4 since we're low on a1 and a4). ", "2. after the pcr oe step (15 cycles on the thermocycler), add 1.25ul of primers 120 and 123 directly to each of the pcr tubes (still in the thermocycler) and run another 20 cycles. ", "If the e. coli containing the successful pcambia-sgrna construct was innoculated into liquid culture last night, miniprep it. if not, lets innoculate more from the patch plate for minipreps tomorrow. ", "Digest ligation d (ask patrick where this is) and pcambia with spei. digest 300ng of each. run both of these next to one another on a gel once finished. "], "Jun22": ["1. re-do pcr of pcocas9 using new primers. try using the gradient options to troubleshoot better melting temperature", "2. run gel of above pcr product. use 3ul of product, 2ul milliq, and 1ul loading dye. ", "3. if every pcr product produces multiple bands, run another gel using 5ul of product and 1ul of dye for gel extraction (add guanosine and 10ul of ladder!). remove 5-6kb band. ", "4. now do the same pcr as the first, but this time on the gel extracted product. run pcr product on a gel and hopefully this time there is just that one, bright 5-6kb band. ", "If the e. coli containing the successful pcambia-sgrna construct was innoculated into liquid culture last night, miniprep it. if not, lets innoculate more from the patch plate for minipreps tomorrow. ", "1. gel extraction of sgrna 123/234 (ask patrick why it's called this and where to get it :p), followed by pcr oe with sgrna 1 and 4 (use c1 and c4 since we're low on a1 and a4). ", "2. after the pcr oe step (15 cycles on the thermocycler), add 1.25ul of primers 120 and 123 directly to each of the pcr tubes (still in the thermocycler) and run another 20 cycles. ", "Make up more km lb agar plates (and just any other agar plates that we're running low on). ", "Calculate transformation efficiency of dh5alpha competent cells.", "Make competent dh5alpha cells."], "Jun20": ["Autoclave the baffled flasks to get them ready for making agrobacterium subculturing", "Test transformation of dh5-alpha competent cells using puc19", "Subculture agrobacterium and place them in moffat's incubator (before noon)", "Take freshly autoclaved 1.5ml tubes out of charles' lab oven.", "Pcr amplify pcocas9 using new primers if they arrive. ", "Ligation of pcocas9 into pcambia, attempt #2. before ligation, run samples to be ligated on a gel to ensure quality. ", "Gel extraction of sgrna 123/234 (ask patrick why it's called this :p), followed by pcr oe with sgrna 1/4 (use c1 and c4 since we're low on a1 and a4). ", "Run diagnostic gel for pcamb+sgrna-a2."], "Jun19": ["Miniprep the inoculated tubes with pcambia-cas9 transformation colonies (10 tubes)", "Miniprep the inoculated tubes with a.tumefaciens-sgrna-a2 colonies. (5 tubes)", "Cp and nano the above two (15 in total)", "Order the sgrna swap sequences and primers?", "Run diagnostic gel for both the miniprepped samples (check benchling for which re to use)"], "Jun18": ["Autoclaved 10ul tips should be dry (sitting in the steamer in the charles lab)", "We need to stock up our lb broth and lb agar. also need to make more km plates (ask peivand about [km]).", "Transformation plates (pcocas9 + pcambia) checked and colonies picked. ", "1. gel extraction of column-purified sgrna 1,2,3,4. use 10ul of sample, and 1.6ul of loading dye. run two lanes of that. don't forget to add a pinch of guanosine! ", "2. cut out bands at 2.5kb (make sure to contact steven before you do this!). nanodrop the resulting product. ", "3. pcr using the gel extract as template. primers 120 and 123. q5. melting temperature 60c. extension time = 1 minute 15 seconds. (double check that those parameters make sense)", "4. run a gel of above pcr before and after column purification of the product. it would be *really* great if we got a single band at ~ 2.5kb after this. cookies for all if successful. ", "Make ampicillin stock (microfuge size), and ran potency test for both the new and old ampicillin powder", "Tranform puc19 into new dh5alpha w/ negative control", "Made 10 amp plates.", "Autoclaved the kimble flask from charle's lab.", "Lab maintenance (garbage, recycling, sweeping, ,sink lab bench/office table wipe down)"], "Jun17": ["Making dh5-alpha competent cells (startting at 7 am) ", "Colony pcr of pcambia in agrobacterium (pick 2 colonies touch plate first then into 50ul) ask patrick use temperature gradient 2 samples", "Transform sgrna-a2+pcambia (cut x/p) into e.coli use positive control (pcambia miniprep 766c+dh5-alpha) and negative control(dh5-alpha only) [total of 4tubes]", "1. digest cas9re with psti followed by column purification. please label this new tube well (date!) and store somewhere safe. ", "2. while digestion of above is happening, run several samples of the many psti-cut pcambia that we have on a gel. (can also run sgrna 1,2,3,4 reaction from last night on this gel)", "3. ligation of the psti-digested cas9re with the best psti-cut pcambia (as determined from gel). don't forget negative control (just pcambia).", "4. after at least 4 hours of incubation at room temperature, transform into dh5-alpha.", "Run pcr oe of sgrna a1,2,3,4 on a gel. load 5ul of each sample and 1ul of loading dye. the three tubes should still be in the thermocycler (were there overnight). ", "Innoculate agrobacterium in different dilutions in room temperature for tomorrow", "Run a diagnostic gel to check colony pcr", "Stuff 5000 tips into boxes...", "Pcr amplify a1,2,3,4 with 120 and 123"], "Jun16": ["Running a gel for cas9re pcr", "Ligate sgrnaa2(cut x/p) with pcambia cut with (x/p)", "Prepare solutions for making dh5-alpha competent cells", "Run autoclave for media and solutions for comp cells ", "Innoculate from dh5-alpha streak plate for tomorrow ", "Gradient pcr of a1+a2 & a3+a4.", "Look into colony pcr protocols and try one for 4 more colonies"], "Jun15": ["Make a 0.8% gel and run the agrobacterium colony pcr tubes on it. there are on the pink rack at 4degree. load 1.5ul of the sample+3.5 water+1ul dye", "Making fresh lb in 1l flask for dh5-alpha competent cells and autoclave at 2pm", "Pcr cas9re using phusion and 100ng of dna in 50ul reaction) this will be just the repeat of what lauren and i did and worked at least for 2-3 times. using 112,113 primers", "Oepcr of a1+a2, b1+b2, c1+c2, followed by addition of primers for extra 20 cycles on top of initial 15 from before.", "Miniprepped more pco_cas9, cp & nano", "Run a diagnostic gel for the oepcr products (cp vs non-cp)", "Run a diagnostic gel for pcr cas9re products (no cp)", "Cleaned test tubes"], "Jun16": ["Pcr of pcocas9 using primers 112 and 113 and tm of 64 degrees. use q5 polymerase. ", "Run a diagnostic gel of the pcr product from above: 2ul pcr product, 3ul water, 1ul loading dye.", "If the gel above produces a single band between 4kb and 5.5kb, column purify the entire rest of the pcr product.", "Run a pst1 digestion on as much dna from the column purified pcr product as possible. column purify after the digestion and you're done!", "Digest 1000ng of pcambia with pst1, then column purify, then nanodrop. "], "Jun12": ["Pcr overlap extension of (a3+a4) with a2. ask for more details from steven. ", "Colony pcr of agrobacterium using cherry's primers", "Pcr of pcocas9 using primers 112 and 113 and tm of 66 degrees. use q5 polymerase. "], "Jun11": ["Time", "Miniprep and test digest the target", "Miniprep and test digest of pcambia", "Make yep media and autoclave", "Test digest of all the ecori enzymes we have", "Pcr repeat of cas9re using primers 112 and 113", "Kfc party"], "Jun10": ["Did you innoculate the target? if yes miniprep and run a diagnostic if no innoculate", "Pcr repeat of cas9re using primers 112 and 113 ", "Test ecori enzyme for contamination"], "Jun9": ["Miniprep of pcambia", "Test digest of pcambia with ecori and diagnostic gel", "Transformation of pcambia into agrobacterium using electroporation", "Transformation of pcambia into dh5-alpha using heat shock (different dna amounts) ", "Did you innoculate or restreak the target results?"], "Jun8": ["Make frozen stock and streak plate of agrobacterium", "Miniprep of pcambia 766 (tube 1 & 2) (incubating)", "Inoculate agro from streak plates and make them plant-competent", "If transformation of targets works, then streak purify", "Update the wiki.", "Make more km plate", "If transformation of cas9+pcambia works, then streak purify", "Make yep liquid media", "Make yep plates (with antibiotics, rif and gentamycin)", "Transformt the cas9re(psti)+pcambia(psti) ", "Transform the target ligations (x12)", "Make frozen stocks and streak of agrobacterium", "Innoculate pcambia 766, 312 on our strain list", "Make lb-agar (and km plates and just lb)", "Cut pcambia with ecori trying different times, 30min,1hr,2,3,4hr and run a diagnostic", "Run oepcr with c2 and c3", "Run diagnostic for the oepcr to check for 1200bp band"], "Jun4": ["Miniprep and digest cas9re+766, pcambia control (psti and sali)", "Run a diagnostic gel on digested cas9re and pcambia", "Make yep media (liquid and plates) ", "Diagnostic digest on pcambia with ecori", "Transform the target ligations (from friday - they are in freezer)", "Digest pcambia with psti and sali and column purify", "Pcr cas9re with psti and sali primers, run on gel, gel extract ", "Digest cas9re with psti and sali and column purify", "Ligate cas9re and pcambia o/n", "Date sunday june7", "Task sets/experiments", "Innoculate the cas9re+766 (from streak purify)", "Streak purify cas9re+766 ligations again", "Make more km plates", "Task sets/ experiments", "Transform the ligation products from yesterday. make sure to create the necessary controls. ", "Run a diagnostic gel to check if the ligation worked. we didn't do this last time so let's do it right :3 (cut w/ psti)", "Autoclave the pippette tips, given that the autoclave comes back to life (broke down last night)", "Digest new pcambia 766 with psti and sali and column puirify (from yesterday", "Ligate pcambia cut psti/sali with cas9-re already digested and column puirifiied from thursday", "Digest pcambia with ecori and run a diagnostic. let it digest for at least two hours. ", "Transform pcambia using heat shock. 50ng of miniprep", "Transform pcambia 766 using electroporation. ", "Make frozen stocks and frozen stock back ups of pcambia 652 (will recieve tomorrow), and streak puirify and miniprep", "Cut pcambia 652 using ecori for 2hours and run a diagnostic. do it parallel with task #8", "Over extension pcr? where are we on that? shall we get samples ready for sequencing a1+a2 and a3+a4?"], "Jun3": ["Task sets/ experiments", "Digest pco-cas9 re using psti and sali and column purify (from yesterday)", "Digest new pcambia 766 with psti and sali and column puirify (from yesterday)", "Ligate #3 and #2 ", "Create 20 lb + km plates", "If target transformation worked, assay(innoculate) colonies", "Digest 284 with xbai and psti + gel extract 1kb/2kb fragments + column purify + nano", "Digest 326 with spei and psti and column puirify", "Digest 332 with spei and psti and column purify", "Ran diagnostic gel with 332 and 326 (expecting 3.5kb and 4kb bands)", "Perform ligation of all the different fragments into 332 and 326", "Filled more pippette tip boxes cause you can never be overstocked with pippette tips...", "Oepcr'd a1-a2", "D o n e .", "Returned media bottles to charles lab"], "Jun2": ["Task sets/ experiments", "Pcr amplify pco-cas9 using primers 112 and 113. use 100ng of dna", "Gel extract the pco-cas9 ", "#6 - digest pco-cas9 re using psti and sali and column purify ", "#7 - digest new pcambia 766 with psti and sali and column puirify ", "Ligate #7 and #6 ", "Run pcr amplified a1+a2 and a3+a4 on a gel ", "Cut new pcambia with ecori (tango, fermentes) and run a diagnostic gel ", "Transform the target construct (make sure to heat shock for only 45 seconds, use controls for 332),patrick,d o n e .,", "Clean test-tubes (the most important task) (please take me)"], "May26": ["Task sets/experiments", "Fill out pippette tip boxes (mostly 0.1-10ul) check others", "Autoclave??"], "Jun1": ["Continuation #6, digest pco-cas9re+digest pcambia", "Continuation on #7 ligate digested pcocas9re into pcambia", "If colonies on target transformation, innoculate some. (#of colonies will be decided with reference to the ligation control)", "Make frozen stock+back up stock+patch plate of new pcambia (#766)", "Digest 284 with xbai and psti and gel extract at 1kb band", "Digest 326 with spei and psti", "Ligate step#11 and 12", "Dilute primers", "Pcr amplify piece 1+2 using primers 120 and 121 followed by coloumn puirification", "Pcr amplify pieces 3+4 using primers 122+123 followed by column puirification "], "May26": ["Experiments"], "May31": ["Wash test tubes & rinse with di", "If colonies grew from 284+332 transformation, colonies need to be innoculated", "Make km plates", "Transform new ligation reaction for target construction using all the controls.", "Cut 284 with ecori and run a diagnostic gel", "Fill up 10ul tip boxes"], "May30": ["Experiments", "Transform ligation reaction 284+332 and 284+326", "Soak test tubes in water and soap"], "May29": ["Experiments", "Miniprep 284, 332", "Digest 284, 332 (column puirify 332) ", "Column puirify 284 ", "Digest and coloumn puirify 284", "Ligate gel extracte 284 and column puirified 332"], }