1. pSK/muFc vector (obtained from the Epstein laboratory).
2. muFc primers: FWD muFc:
5'CCCTATAAGCTTCGrACGGAGCCCAGAGGGCCCACAATC (HindIII site; BsiWI site; Hinge region) REV muFc:
5'ATCAATGAATTCTCACGCGGCCGCTTTACCCGGAGTCCGGGAGAA (£coRI site; NotI site; CH3 region)
3. pEE12 GS expression vector (NS0 cells included; Lonza Biologics).
4. HindIII restriction enzyme (RE) (10X NEBuffer 2 included; NEB, cat. no. R0104S).
5. £coRI RE (10X NEBuffer £coRI included; NEB, cat. no. R0101S).
6. Montage PCR filter units (Millipore, cat. no. UFC7PCR50).
7. T4 DNA ligase (10X ligation buffer provided; NEB, cat. no. M0202S).
8. XL1-Blue Escherichia coli bacteria (Stratagene, cat. no. 200268).
10. Bacterial shaker capable of 150 rpm.
11. Luria broth (LB), prepared according to manufacturer (Sigma, cat. no. L7658).
12. Ampicillin (Amp; Sigma, cat. no. A9518): prepare 25 mg/mL stock in sterile dH2O.
13. LB plus Amp (LB/Amp): dilute Amp stock 1:250 in sterile LB.
14. LB agar plates plus Amp 100 p,g/mL, prepared according to manufacturer (Sigma, cat. no. L7533).
15. Bacterial incubator, 37°C, nonhumidified.
16. Polypropylene round-bottom 14-mL tubes with caps (BD Falcon, cat. no. 352059).
17. Toothpicks, wooden, autoclaved.
18. Wizard Plus SV Miniprep kit (Promega, cat. no. A1330).
19. pSK/muFc vector map file (obtained via email from the Epstein laboratory).
Was this article helpful?