By Choi Sang Long

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6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. I-SAGE/I-LongSAGE kit with magnetic stand (Invitrogen, Carlsbad, CA). 5-mL tubes (Ambion, Inc, Austin, TX). , Lake Success, NY). , Beverly, MA). T4 DNA ligase (5 U/ L) from USB (Cleveland, OH). Polyacrylamide gel electrophoresis (PAGE)-purified oligos (Integrated DNA Technologies Inc, Coralville, IA) (8). Linker 1A: 5 -TTTGGATTTGCTGGTGCAGTACAACTAGGCTTAATATCCGACATG-3 Linker 1B: 5 -TCGGATATTAAGCCTAGTTGTACTGCACCAGCAAATCC-C7 amino-modified-3 Linker 2A: 5 -TTTCTGCTCGAATTCAAGCTTCTAACGATGTACGTCCGACATG-3 Linker 2B: 5 -TCGGACGTACATCGTTAGAAGCTTGAATTCGAGCAG-C7 amino-modified-3 PCR primers (7–8): Primer 1: 5 -biotin GTGCTCGTGGGATTTGCTGGTGCAGTACA-3 Primer 2: 5 -biotin GAGCTCGTGCTGCTCGAATTCAAGCTTCT-3 Magnetic stand (Invitrogen, Carlsbad, CA).

2. Electroporation device. 3. Luria-Bertani (LB) broth (Sigma, Taufkirchen, Germany). 4. Zeocin (100 mg/mL; Invitrogen) (store at −20 C, this antibiotic is light-sensitive). 5. 5-Brom-4-chlor-3-indoxyl- -d-galactoside (X-Gal) (Roth, store at −20 C). 6. 5 % (w/v) agar in LB broth. 12. Insert-PCR 1. 5 mg/mL BSA. 2. 50 mM magnesium chloride solution. 3. Oligonucleotides: Insert_for: 5 -CTG GTT AAC CTT ACT GGC TGA GTT AGC TCA CTC ATT AGG CAC-3 Insert_rev: 5 -TGT AAA ACG ACG GCC AGT TAC GAC TCA CTA TAG GGC GAA TTG-3 .

Incubate for 2–3 h at 16 C. Inactivate ligase by incubation for 30 min at 70 C. 5. Add 10 L sterile MilliQ water to the control reactions. Ligation reactions can be stored at −80 C. 6. Add 80 L LoTE to each of the ligation reactions, and 100 L PCI and mix vigorously (see Note 16). 7. Centrifuge at maximum speed in a benchtop centrifuge for 5 min and transfer upper aqueous phase to a new tube. 8. 5 M ammonium acetate, 3 L glycogen and 500 L 100 % ethanol. Mix well. 9. Incubate at −40 C for 30 min and centrifuge at maximum speed in a benchtop centrifuge and 4 C for 30 min.

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