1. PCR amplify the barcoded insert off of Lenti-PGK/Cre plasmid.
  
Reaction mix:
200 l PrimeStarHS Mix
12 l  Forward Primer \(10 M)
12 l  Reverse Primer \(10 M)
8 l Template \(500 ng/L)
168 l H20                          
--------------------------------------
400 l Total  Split into 4, 100 l reactions
PCR Program:
   Temp. \(°C)  Time \(min)
1. 98              1:00
  2. 98              0:15
  3. 55              0:05
  4. 72              1:00
  5. Go to step 2, 34x
  6. 72              5:00
  7. 4                forever
  
2. Pool the PCR reactions and PCR purify using the Qiagen® PCR Purification Kit. Elute in 35 l H20. 
  3. Digest barcoded insert with BspEI and BamHI.
  Reaction mix:
5 l Buffer 3.1
1.5 l  BspEI
1.5 l  BamHI
3 g Barcode Insert 
H20 to 50 l                         
--------------------------------------
50 l Total  Incubate at 37°C 2 hours
4. Concurrently digest Lentiviral Plasmid with XmaI and BamHI.
  Reaction mix:
5 l Cutsmart Buffer 
1.5 l  XmaI
1.5 l  BamHI
5 g Lentiviral Plasmid
H20 to 50 l                         
--------------------------------------
50 l Total  Incubate at 37°C 2 hours
5. Run out digested products on a 2% agarose gel and gel extract the fragments using the Qiagen® MinElute Gel Extraction Kit \(Qiagen, 28604) and elute in 25 l of H20.
  6. Ligate digested barcoded insert into digested Lentiviral Plasmid
  Reaction mix:
8 l   Ligation Buffer 
1.6 l   T4 DNA Ligase \(NEB, M0202)
300 ng Digested Lentiviral Plasmid
180 ng Digested Barcoded Insert 
H20 to 80 l                         
--------------------------------------
80 l Total  Incubate at 16°C overnight
7. PCR purify the ligation product using the Qiagen® PCR Purification Kit \(Qiagen, 28106)
  8. Transform 1 l into 20 l ElectroMAX DH10B cells \(Thermo Fisher, 18290015). Use 0.1 cm GenePulser/MicroPulser Cuvettes \(Bio-Rad, 165-2089) and BD MicroPulser™ Electroporator \(Bio-Rad,165-2100)at 1.9 kV. 9. Ad 500 l of SOC media and shake at 200 rpm for 30 minutes at 37°C.
  10. Plate on LB-Amp plates \(1x1 l, 1x 10 l, and 5x 100 l) and incubate at 37°C overnight.
  11. Count colonies on either the 1 l or 10 l plate and use these counts to estimate the colony number on the 100 l plates. The total number of colonies counted will provide an estimate of the number of unique barcodes obtained.
  12. Scrape bacterial colonies off of the LB-Amp plates in 10 mL of LB-Amp per plate.
  13. Pool liquid in a flask and shake at 200 rpm for 30 minutes at 37°C.
  14. Midi-prep the barcoded plasmid pool using the Qiagen® HiSpeed MidiPrep Kit \(12643).
  15. Determine plasmid concentration and confirm barcoding by Sanger sequencing.