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Design and Application of a shRNA 213Fig. 1. Restriction map of pPUR V2 vector. The original BamHI and EcoRI sites inpPUR have been destroyed. The U6-shRNA cassette, generated by PCR, is subclonedinto the EcoRI and XhoI sites of pPUR V2.4. 2 U/µL Deep vent DNA polymerase (New England Biolabs [NEB: Ipswich, MA]).5. Qiaquick PCR purification kit (Qiagen).6. Restriction enzymes and buffers: EcoRI and XhoI (NEB).7. 10X Tris-Acetate-EDTA (TAE) agarose gel-loading dye (150 mM ethylenediaminetetraaceticacid, 30% glycerol, 0.25% [w/v] bromophenol blue [Sigma, St.Louis, MA], and 0.25% [w/v] xylene cyanol FF [Sigma]).8. 1% UltraPure agarose (Invitrogen).9. Low melting temperature agarose (SeaPlaque Agarose, Cambrex: Walkersville, MD).10. T4 DNA ligase (Roche: Basel, Switzerland).11. DH5α competent cells (Invitrogen).12. Qiafilter plasmid Maxi kit (Qiagen: Valencia, CA).13. Laemmli sample buffer (50 mM Tris-HCl, 2% [w/v] sodium dodecyl sulfate[SDS], 100 mM dithiothreitol, 10% [v/v] glycerol, and 0.05% [w/v] bromophenolblue, pH 6.8).14. Equipment and reagents for SDS-polyacrylamide gel electrophoresis (PAGE).15. Bradford reagent: Bio-rad, Hercules, CA and 1 mg/mL bovine serum albuminas standard.16. Polyvinylidene (PVDF) protein transfer membrane (Bio-Rad: Hercules, CA).17. Towbin transfer buffer (170 mM glycine, 22 mM Tris-HCl, and 0.01% [w/v] SDS,pH 8.3).