Target Discovery and Validation Reviews and Protocols

146 Sano and Taira
5. 10X L (low-salt) buffer: 100 mM Tris-HCl, pH 7.5, 100 mM MgCl 2 , 10 mM
dithiothreitol (DTT).
6. 5 U/µL Taq DNA polymerase.
7. Restriction enzymes: 8 U/µL Csp45I; 10 U/µL KpnI.
8. 50X TAE buffer: 2 M Tris-acetate, 0.05 M EDTA, pH 8.0.
9. Ethanol: both 100 and 70% in distilled water.
10. Phenol and chloroform.
11. Agarose.
12. SOC medium: 10 g bacto-tryptone, 5 g bacto-yeast extract, 10 g NaCl in 1 L
distilled water.
13. Escherichia coli (JM109, DH-5α or other) competent cells.
14. Kit for purification of DNA fragments from agarose gels (e.g., QIAquick Gel
Extraction Kit; Qiagen, Hilden, Germany).
15. Kit for purification of PCR products (e.g., QIAquick PCR purification kit; Qiagen).
16. Kit for ligation of DNA fragments to the vector (e.g., DNA Ligation kit Ver.2;
Takara Shuzo Co., Kyoto, Japan).
2.2. Introduction of a Ribozyme Library Into Cells
and Recovery of the Ribozymes
1. Cell lines (e.g., HeLa S3, HT1080, B16-BL6 mouse melanoma cells or other).
2. Cell growth medium (e.g., Dulbecco’s modified Eagle’s medium [DMEM]; RPMI
1640, Sigma Chemical, St. Louis, MO).
3. Serum-free medium (e.g., Opti-MEM I, Life Technologies).
4. Fetal bovine serum.
5. Antibiotic mixture (e.g., Antibiotic-Antimycotic, Life Technologies).
6. Transfection reagents (e.g., TransIT-LT1, Pan Vera, Madison, WI; LipofectAMINE 2000, Invitrogen, Carlsbad, CA).
7. PBS buffer: 8000 mg NaCl, 200 mg KCl, 1150 mg Na2HPO4 (anhydrous), 200 mg
KH2PO4 (anhydrous) in 1000 mL distilled water.
8. Trypsine-EDTA.
3. Methods
3.1. Construction of a Library Expressing Randomized
Hammerhead Ribozymes
The method for constructing a hammerhead ribozyme library includes
three steps. At first, DNA templates encoding ribozyme catalytic core flanked
by a substrate-binding region that is completely randomized should be chemically
synthesized and amplified by polymerase chain reaction (PCR). After
restriction digestion of PCR fragments, they are ligated into the plasmid.
Finally, the plasmids are used for transformation of competent E. coli cells.
Importantly, we must care and maintain the diversity of a ribozyme library in
all steps.