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Map of Experiments for Tumor cDNA Library

2015-01-13

azim58 - Map of Experiments for Tumor cDNA Library * competent cells BL21 1-13-10 * transformation to check BL21s 1-13-10 * transformation results 1-20-10 * 4T1 Cell Culture 5-10-10 to 5-18-10 * Harvesting 4T1 Cells 5-24-10 * Tumor Injection and Genetic Immunization 5-25-10 * Spleen Processing 6-12-10 * Tumor and B cell RNA Extraction 6-14-10 * Tumor, spleen, and blood collection 6-18-10 * First strand tumor cDNA Synthesis 1-20-11 * Gel of optimized PCR 1-21-11 * double strand tumor cdna synthesis 1-21-11 * ds cDNA PCR Optimization 1-24-11 * tumor ds cDNA on TAE Gel 1-26-11 * 2nd tumor cDNA synthesis (no delay after 1st strand) 1-28-11 * 3rd tumor cDNA synthesis (DEPC H20) 1-31-11 * 4th Tumor cDNA synthesis (4 ug instead of 1 ug) 2-1-11 * Gel of tumor ds cDNA 2-2-11 * ds cDNA purification and size fractionation 2-9-11 * gel of purified cDNA 2-11-11 * ligation of tumor cDNA into pSMART2IF 2-16-11 * Tumor cDNA transformation 2-22-11 * In-fusion tumor cDNA transformation results 2-23-11 * tumor cDNA library decision 3-3-11 * transformation results of DH10B w/PUC19 3-3-11 * test electroporation w/0.1 cm cuvette 3-8-11 * SMARTer cDNA synthesis (of 4T1 Tumor RNA) 3-8-11 * Gel of ds tumor cDNA 3-9-11 * Large plating of practice 10^5 tumor cDNA library 3-9-11 * cDNA library calculations 3-9-11 * tumor 4T1 cDNA purification 3-10-11 * In-fusion cloning of tumor cDNA library 3-11-11 * test electroporation with dried plates 3-14-11 * transformation results 3-15-11 * transformation calculations 3-15-11 * plates required for cDNA library 3-17-11 * precipitation of DNA ligation 3-21-11 * library titration calculations 3-21-11 * minimum tumor cDNA library transformation 3-21-11 * minimum tumor cDNA library transformation 2 3-24-11 * colony pcr of tumor cDNA library 3-25-11 * miniprep of tumor cDNA colonies 3-29-11 * follow up sequencing 4-1-11 * Transformation of tumor cDNA library with electroporator 4-13-11 * Transformation of Tumor cDNA w/200 pg 4-14-11 * Transformation of library for printing 5-3-11 * Tumor Library Plate Cultures 5-5-11 * Colony PCR of Tumor Library Cultures 5-6-11 * Gel of Colony PCR 5-9-11 * Tumor library overnight culture 5-9-11 * Extract tumor library proteins 5-10-11 to 5-11-11 * Extract tumor library proteins protocol 5-11-11 * Tumor library array experiment 5-13-11 * Western blot to check for SMC1fs protein 5-17-11 * 2nd half of western blot 5-18-11 * Transform BL21 w/pGEX 5-23-11 * Growth of Culture for Protein Extraction 5-24-11 * Protein Extraction of SMC1fs in Plate Format 5-26-11 * Western Blot of SMC1fs 5-26-11 * Transformed BL21 with P/pGEX &pET32b 6-1-11 * Protein extraction from BL21 6-2-11 * Western blot for SMC1fs 6-3-11 * Western blot results 6-6-11 * Extraction of protein w/Lysozyme &sonication 6-8-11 * Coomassie to Identify 14 KD band 6-13-11 * Test of anti-rabbit for 14 KD band 6-14-11 to 6-16-11 * Protein expression with less lysozyme 6-20-11 * Western blot to test for 14 KD band 6-20-11 * Western blot to test for 14 KD band 6-22-11 * Preparation of Cell lysate plate 6-27-11 * Printing cell lysate 2nd time 6-30-11 * CodeLink cell lysate test 7-8-11 * Cell lysate plate preparation 7-14-11 * Cell lysate slides 7-21-11 * Cell lysate plate preparation 8-2-11 * Running HEPES and Glycerol cell lysate slides 8-8-11 * Cell lysate plate preparation 8-9-11 * Valiery cell lysate slides 8-26-11 * 1000 cfu/100ul overnight culture 8-27-11 * SMC1fs protein expression 9-13-11 * Transform BL21 with pET32-TEV 9-17-11 * Produce protein from pET32-TEV 9-19-11 * Express proteins from pGEX-SMC1fs 9-20-11 * Cell lysate printing 7 9-23-11 * Chemical transformation pGEX-SMC1fs 9-26-11 * Produced SMC1fs protein 9-27-11 * Western blot check for SMC1fs protein 9-30-11 * Protein production of SMC1fs 10-1-11 * Western blot to check for SMC1fs protein 10-3-11 * Western blot image of SMC1fs 10-4-11 * Production of SMC1fs in Assay plates 10-6-11 * SMC1fs cell lysate western blot 10-10-11 * SMC1fs cell lysate check 10-15-11 * Cell lysate nitrocellulose slides with Tecan 10-15-11 * * Cell lysate on nitrocellulose w/o accidental prewash 10-26-11 * Concentrated primary and long incubation time 11-6-11 * Nitrocellulose Grace Biolabs Slides 12-13-11 * Super G Blocking Buffer with nitrocellulose 12-19-11 * 4T1 cDNA library transformation 1-6-12 * 4T1 cDNA Library Protein Production in plate format 1-7-12 * single electroporation 1-9-12 * electroporations 1-10-12 * transformation results 1-11-12 * protein production tumor library 1-11-12 * production of protein 1-12-12 * culture pcr of tumor libraries 1-12-12 * minipreps and pcr for B & C Ligations 1-13-12 * B & C Tumor cDNA Gel 1-17-12 * Sequencing of tumor B & C Ligations 1-18-12 * Transformation results of A tumor ligation 1-18-12 * Protein production 1-19-12 * tumor library transformation results 1-20-12 * electroporate tumor library 1-23-12 * transformation results 1-24-12 * Glycerol Stock Test 1-25-12 * B Tumor cDNA Library Ligations * Transformation results 1-31-12 * produce tumor protein 2-1-12 * HiGro Test 2-3-12 * Ab purification with protein G column 2-3-12 * Time and blocking buffer test 2-11-12 * Check of protein in tumor cDNA library lysate samples 2-13-12 * 28 electroporations 2-15-12 * colony count with ImageJ 2-16-12 * protein production in plate format 2-17-12 * protein production in plate format 2-21-12 * mouse sera on cDNA library arrays 2-29-12 * colony hybridization test 3-12-12 * colony hybridization protocol test 3-15-12 * test of colony hybridization without 250 rpm and freeze/thaw 3-16-12 * tumor library glycerol stock titration 3-18-12 * immunofluorescence with naive sera 3-20-12 - 3-21-12 * 2G2 Protein 3-22-12 * produce protein in large bioassay tray 3-23-12 * tumor cDNA library on bioassay tray 3-26-12 * protein production 3-28-12 * application of antibodies 3-28-12 * colony hybridizations 3-31-12 * pcr of positive smc1fs pools 4-2-12 * bacteria facs test 4-5-12 * pcr of facs sorted cells 4-9-12 * ELISPOT 4-8-12 * sequenced colonies binding to tumor sera 4-13-12 * secondary titration on cDNA expression arrays 4-17-12 * SMC1fs PCR of tumor cDNA Library 5-1-12 * Dilution of SMC1fs Spots 1 to 1000 5-2-12 * tcDNA library with 0.1 nM Secondary 5-4-12 * Cell tracker dye experiment 5-7-12 * sort overnight culture with FACS 5-8-12 * E coli lysate investigation 5-17-12 * glycerol and sodium azide for preservation 5-18-12 * CpG, GST, and E coli ELISA 5-18-12 - 5-21-12 o Repeat of ELISA with correct plate 5-22-12 * Removal of anti-adjuvant antibodies 5-29-12 to 5-30-12 * Secondary titration experiment 6-6-12 * transfer lysate for printing (spot validation test) 6-9-12 * preparation for sera purification 6-11-12 * sera purification with random peptides 6-12-12 * Dilution of culture 6-19-12 * protein production 6-21-12 * Continuation of protein production 6-25-12 * Counting Tentagel Beads for Density 6-28-12 * Sequencing Tumor Library Colonies 7-10-12 * Produce selected tumor library proteins 7-18-12 * Breast tumor RNA Purity Check 7-25-12 * Naive and tumor sera on cell lysate array 7-27-12 * Glycerol stock of selected 16E3 wells 8-9-12 * selected 16E3 plasmid transformation 8-27-12 * protein production for printing 8-30-12 * cuvette cleaning test 8-30-12 * tumor and naive sera on selected 16E3 lysate 9-7-12 * tumor and naive sera at 1:100 on selected 16E3 lysate 9-14-12 * dscDNA Purification 9-28-12 * Tumor Lib lysate arrays 3 Dilutions 9-29-12 * electroporate tumor cDNA library 10-9-12 * Library colony count 10-12-12 * 1:2000 dilution on lib lysate arrays 10-13-12 * tumor cDNA purification 10-17-12 * Gel of purified DNA 10-17-12 * transformation of human cDNA 10-30-12 * human library transformatrion at higher concentration 10-29-12 * checked tumor library transformation 10-31-12 * collected more human tumor cDNA aliquots 11-1-12 * Tumor cDNA purification 10-17-12 * Gel of purified DNA 10-17-12 * Transformation of human cDNA 10-30-12 * Human library transformation at higher concentration 10-29-12 * checked tumor library transformation 10-31-12 * collected more human tumor cDNA aliquots 11-1-12 * in-fusion of tumor cDNA 11-2-12 * transformation efficiency check 11-3-12 * transformation w/1ng DNA 11-6-12 * transformation results 11-7-12 * test of electroporation parameters 11-7-12 * tumor library pcr screen 11-9-12 * tumor library sequencing 11-13-12 * cDNA PCR Screen 11-27-12 * beta actin pcr screen 11-27-12 * cDNA PCR Screen 11-29-12 * cDNA Synthesis Kit Test 11-30-12 * PCR Screen with Switched Kits 12-1-12 * cDNA library PCR technical repeat 12-17-12 * tumor cDNA random hex 1st strand synthesis 1-24-13 * random hexamer 2nd strand synthesis 1-25-13 * 2nd strand synthesis with more cycles 1-28-13 * cDNA size fractionation 1-30-13 * planned to amplify specific mRNA transcripts 2-1-13 * ethanol precipitation of random hex lib 2-3-13 * transformation results 2-4-13 * control in-fusions for test 2-4-13 * miniprep tumor library cultures 2-5-13 * transformation efficiencies 2-6-13 * amplify specific RNA transcripts 2-6-13 * 2nd strand synthesis 2-7-13 * transcript PCR gel images 2-7-13 * transcript DNA for blunt end cloning 2-11-13 * PCR purification 2-12-13 * Amresco/Seakem agarose comparison 2-14-13 * RNA integrity check 2-19-13 * gel electropheresis optimization 2-20-13 * RNA check at different voltages 2-21-13 * RNA gel 70 V 1 hr * last check of RNA 2-25-13 * 2nd strand synthesis 2-27-13 * dynazyme reaction 2-27-13 * 5 additional cycles 2-28-13 * ampure magnetic bead purification 3-1-13 * in-fusion reaction 3-7-13 * pcomb miniprep 3-19-13 * condition test for RNA transcript amplification 3-19-13 * PCR on optimization of RT-PR Rxns 3-20-13 * Electroelution practice 3-22-13 * Gel of electroeluted samples 3-23-13 * Pentadecamer library synthesis 3-23-13 * Pentadecamer dynazyme 3-24-13 * specific transcript amplification 3-24-13 * PCR optimization 3-25-13 * reamplify pentadecamer library 3-26-13 * electroelution 3-28-13 * 5 more cycles for transcript specific amplification 3-31-13 * transcript specific amplification 4-1-13 * investigate pComb sequencing problems 4-2-13 * transcript specific amplification 4-4-13 * tumor pool screen 4-5-13 * transcript amplification 4-5-13 * tumor pool screen 4-8-13 * miniprep of tumor lib pools 4-9-13 * started ethanol precipitation of electroeluted DNA 4-10-13 * tumor library transcript amp 4-12-13 * SMC1fs transcript amp 4-14-13 * SMC1fs screen 0.3 uM final primer 4-15-13 * SMC1fs PCR with pools 4-16-13 * Pentadecamer Library In-fusion 4-16-13 * dynazyme and primestar SMC1fs test 4-17-13 to 4-18-13 * ethanol precipitation of in-fused dna 4-19-13 * test of touchdown technique 4-20-13 * smc1fs pcr 4-20-13 * electroporation results 4-20-13 * beta-actin pcr test 4-22-13 * smc1fs pcr 4-22-13 * smc1fs screen of pools 4-23-13 * touchdown for eatr and rs8 4-23-13 * pcomb sequencing 4-23-13 * smc1fs pool screen with touchdown 4-24-13 to 4-26-13 * smc1fs screen for 21-41 pools 4-28-13 * smc1fs screen of 2-17-12_15 plate 5-1-13 * smc1fs screen of 2-17-12_15 plate 5-1-13 * repeat of 2-17-12_15 screen 5-2-13 * 13 chimeric transcript screen 5-2-13 * 2-17-12_15 SMC1fs screen with tubes 5-3-13 * eatr and rs8 with dynazyme hot start method 5-7-13 * electroporation test 5-8-13 * transformation results 5-8-13 * specific transcript PCR with multiple extension times 5-9-13 * PCR condition test for Eatr and RS8 5-10-13 * Gel of Eatr and RS8 5-12-13 * small primer start with touchdown 5-14-13 * Eatr PCR troubleshooting 5-16-13 * SMC1fs control and validation pcr 5-16-13 * SMC1fs PCR contamination test 5-16-13 * SMC1fs screen for pools 1 & 8 5-20-13 * "PCR screen of 41 pools for 7 chimeric transcripts" and "96 ovnt cultures from 1-26-12_2" 5-21-13 * 96 SMC1fs PCRs for 1-26-12_2 plate with dynazyme 5-28-13 * SMC1fs PCR for selected samples dynazyme 5-30-13 to 5-31-13 * Validate chimeric transcripts with dynazyme 5-31-13 * Chimeric transcript screen key 5-31-13 * Screened SMC1fs with 10 pools with dynazyme 6-10-13 * pJET cloning rxn with 4 SMC1fs gel extracted fragments 6-14-13 * transformation efficiencies obtained 6-16-13 * chimeric transcript validation pcr for selected tubes 6-17-13 to 6-18-13 * SMC1fs PCR for orientation 6-20-13 * Perform SMC1fs screen with 6 6-10-13 * transcript pcr optiization with Eatr 6-21-13 * gel extracted bands from buffer in fridge 6-22-13 * clone gel extracted fragments into pJET 6-24-13 * transcript validation with small primer start touchdown 6-24-13 * SMC1fs bands 6-25-13 * Eatr gel 6-25-13 * PCR with pJET sequencing primers on minipreps 6-26-13 * larger volume of SMC1fs for gel extraction 6-27-13 * slain and rbm screen on 1-41 pools 6-27-13 * Amlify both eatr and rs8 6-28-13 * smc1fs pcr with 87 and 6 6-28-13 * slain and rbm transcripts 6-28-13 * eatr and rs8 gel 6-28-13 * check of smc1fs conditions 6-29-13 to 6-30-13 * smc pcr with old and new dNTPs 7-1-13 to 7-2-13 * chimeric transcript amplification for sera 7-2-13 * slain and rbm pcr with primestar max 7-3-13 * 1st strand cDNA synthesis 7-8-13 * test of cDNA quality 7-9-13 * screen of 41 pools with slain primers 7-10-13 * smc1fs pcr with 87 and 6 for gel extraction 7-11-13 * results from 41 pool slain screen 7-11-13 * smc gel with 6 and 87 7-11-13 * loaded larger volume of smc sample 7-12-13 * smc pcr with primestar 7-12-13 * clone gel extracted fragments into pJET 7-14-13 * chimeric transcript antibody pcr 7-15-13 * screen for smc1fs, smc_fus, and slain 7-18-13 * amplify smc_fus for sequencing 7-23-13 * screen 96 pools in 16 for slain 7-23-13 * sequenced potential smc_fragments 7-25-13 * nested pcr for smc_fus and slain_fus 7-29-13 * test of plasmid pool concentrations with pcr 7-30-13 * nested pcr with 87 with 10 pg 7-31-13 to 8-1-13 * pcr with 87 with dynazyme 8-1-13 to 8-2-13 * pcr with 87 with dynazyme 8-2-13 - smc nested pcr 8-5-13 - smc nested pcr with fresh reagents 8-6-13 - miniprep of 96 1-26-12_2 cultures 8-8-13 - smc nested pcr w/fresh miniprep 8-9-13 - smc pcr 8-13-13 - smc pcr 8-13-13 - cbx3 pcr 8-14-13 - cbx3 nested pcr 8-20-13 - dna sequencing key 8-20-13 - smc 87 pcr 8-22-13 - clone cbx3 fragment 8-23-13 - miniprep & pcr of cbx3 8-26-13 - nested pcr of cbx3 8-28-13 - sequence unknown product from cbx3 pcr 9-9-13 - nested pcrs for specific transcripts 9-9-13 - cloned extra pcr fragments 10-3-13
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