By N.J. Clifton
Read Online or Download Cardiac Cell and Gene Transfer: Principles, Protocols, and Applications (Methods in Molecular Biology Vol 219) PDF
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Extra info for Cardiac Cell and Gene Transfer: Principles, Protocols, and Applications (Methods in Molecular Biology Vol 219)
7. 22-µm filter sterilized. 8. CaCl2, 2 M. 9. Chloroquine, 100 mM. 10. 4 (Gibco-BRL). 11. 45-µm filter sterilized. 12. 10% NP-40 in sterile water. 13. 250-mL Centrifuge bottles (Kendro Laboratory Products, Newtown, CT). 14. 45 µm filtered). 15. Cell scraper (Sarstedt, Newton, NC). 16. DNase I (10 mg/mL). 17. RNase A (10 mg/mL). 18. 0, 1 mM MgCl2. 19. 45 µm filtered). 20. 45 µm filtered). 21. 45 µm filtered). 22. CsCl (powder). 23. Dialysis membrane or cassettes (10,000 mw cutoff; Slide-a-Lyzer, Pierce, Rockford, IL).
High-fidelity polymerase chain reaction (PCR) kit. Taq DNA polymerase has a relatively high error rate. To decrease PCR-related mutations, we recommend using the Expand High Fidelity PCR System from Roche (Indianapolis, IN, #1-732641). The 5'-3' exonuclease proofreading activity of the Pwo DNA polymerase in this system results in DNA synthesis with a much higher fidelity. Store at –20°C. (see Note 4). 2. Electroporation-competent E. coli SURE cells (>5 × 109 transformants/µg DNA; Stratagene, La Jolla, CA, #200227): Genotype (e14- (McrA-) D(mcrCBhsdSMR-mrr)171 endA1 supE44 thi-1 gyrA96 relA1 lac recB recJ sbcC umuC::Tn5 (Kanr) uvrC [F' proAB lacIqZD(M15 Tn10 (Tetr)]).
7. Glycerol shock the cells by aspirating the medium from the cells and gently washing the monolayer with prewarmed PBS. 5 mL of 15% glycerol/1X HBS solution to the cells. After 40 s, remove the glycerol solution and rinse the cells twice with PBS. 8. Re-feed the plates with fresh DMEM + FBS. Incubate at 37°C until viral CPE reaches 100%, usually 8–12 d (see Note 5). 9. Collect the cells and medium from the plate and freeze/thaw 3 times in a dry iceethanol bath and a 37°C water bath. Store at –70°C.
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