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