PCR amplify the barcoded insert off of Lenti-PGK/Cre plasmid.
{"action": "amplify", "output": "", "reagent": ["the barcoded insert"]}
Go to step 2, 6. #####%%%%% output "the Qiagen® PCR Purification Kit" are used in line6
{"action": "go", "output": "the Qiagen\u00ae PCR Purification Kit", "reagent": [""]}
Pool the PCR reactions using the Qiagen® PCR Purification Kit. #####%%%%% output "Lentiviral Plasmid" are used in line8
{"action": "pool", "output": "Lentiviral Plasmid", "reagent": ["the Qiagen\u00ae PCR Purification Kit"]}
Concurrently digest Lentiviral Plasmid with XmaI . #####%%%%% output "digested products" are used in line10
{"action": "digest", "output": "digested products", "reagent": ["Lentiviral Plasmid", "XmaI", "Lentiviral Plasmid"]}
Run out digested products on a 2% agarose gel . #####%%%%% output "Qiagen® MinElute Gel Extraction Kit (Qiagen, 28604)" are used in line12
{"action": "run", "output": "Qiagen\u00ae MinElute Gel Extraction Kit (Qiagen, 28604)", "reagent": ["digested products"]}
extract the fragments using the Qiagen® MinElute Gel Extraction Kit \(Qiagen, 28604) #####%%%%% output "H20" are used in line14
{"action": "extract", "output": "H20", "reagent": ["Qiagen\u00ae MinElute Gel Extraction Kit (Qiagen, 28604)"]}
elute in 25 l of H20 #####%%%%% output "Lentiviral Plasmid" are used in line16
{"action": "elute", "output": "Lentiviral Plasmid", "reagent": ["H20"], "volume": ["25 l"]}
Ligate digested barcoded insert into digested Lentiviral Plasmid Reaction mix: 8 l Ligation Buffer 1.6 l T4 DNA Ligase \(NEB, M0202) l -------------------------------------- 80 l Total Incubate at 16°C overnight 7.
{"action": "ligate", "output": "", "reagent": ["barcoded insert", "Lentiviral Plasmid", "Ligation Buffer"]}
300 ng Digested Lentiviral Plasmid 180 ng Digested Barcoded Insert H20 to 80 #####%%%%% output "the ligation product" are used in line20
{"action": "read", "output": "the ligation product", "time": [""]}
PCR purify the ligation product using the Qiagen® PCR Purification Kit \(Qiagen, 28106) 8.
{"action": "purify", "output": "", "reagent": ["the ligation product"], "device": ["the Qiagen\u00ae PCR Purification Kit (Qiagen, 28106) 8"]}
Transform 1 l \(Thermo Fisher, 18290015)into 20 l ElectroMAX DH10B cells .
{"action": "transform", "output": "", "reagent": ["Thermo Fisher, 18290015", "ElectroMAX DH10B cells"]}
Use 0.1 cm GenePulser/MicroPulser Cuvettes \(Bio-Rad, 165-2089) 1.9 kV.

The total number of colonies counted will provide an estimate of the number of unique barcodes obtained. #####%%%%% output "colonies" are not used
{"action": "provide", "output": "colonies", "reagent": [""]}
Scrape bacterial colonies off of the LB-Amp plates in 10 mL of LB-Amp per plate.
{"action": "peel", "output": "", "reagent": ["bacterial colonies", "LB-Amp", ""], "container": ["plate"]}
Determine plasmid concentration . #####%%%%% output "Sanger" are used in line32
{"action": "determine", "output": "Sanger", "concentration": ["plasmid concentration"]}
confirm barcoding by Sanger sequencing
{"action": "confirm", "output": "", "reagent": ["Sanger"]}