John M. S. Bartlett.pdf - Bio-Nica.info

504 Ravassard et al.
9. ddH 2 O.
10. Dynabeads mRNA purification kit (Dynal, Lake Success, NY).
2.3. Synthesis of the ss-cDNA
1. Water bath at 70 and 42°C.
2. Dry-ice powder and ice.
3. 10× FSB buffer: 1 M Tris-HCl, pH 8.4 (at 42°C), 1.2 M KCl, 100 mM MgCl 2 . Aliquot
and store at –20°C.
4. DTT (100 mM ).
5. Acetylated bovine serum albumin, 5 mg/mL (RNase-free; Life Technologies/BRL,
Gaithersburg, MD).
6. RNasin, 36 U/µL (Promega Biotech, Madison, WI).
7. dNTPs, 10 mM each (Use lithium-free dNTPs).
8. Sodium pyrophosphate (20 mM; PPi).
9. [α 32 P] dATP, 3000 Ci/mmol (Amersham, Arlington Heights, IL).
10. AMV reverse transcriptase, 10 U/µL (Promega Biotech, Madison, WI).
11. RA3′ NV (50 ng/µL).
2.4. Synthesis Yield Determination
1. DE81 Whatman paper.
2. Na 2 HPO 4 (0.5 M ).
3. 100% Ethanol.
4. Aqueous scintillation cocktail.
2.5. Removal of Primers
1. Prep-A-Gene DNA purification Kit (Bio-Rad [Richmond, CA]; #732-6010). The silica
matrix used in this kit does not bind RNA or small DNA molecules (cut off around 100
nucleotides) under oxidizing conditions.
2. Water baths at 90 and 65°C.
2.6. Ligation of the ss-cDNA to the Modified Oligonucleotide
Modified oligonucleotide: 5′ phosphate and 3′ NH 2 A5′NV (or B5′NV).
2.7. PCR Amplification
PCR reagents, including primers.
2.8. Direct Screening of the PCR Library
with Biotinylated Oligonucleotides
1. Biotinylated primer (see Subheading 2.1.2.).
2. Streptavidin Dynabeads (10 mg/mL Dynabeads M-280 Streptavidin, DYNAL, Lake
Success, NY).
3. Magnetic concentrator (Dynal MPC).
4. Rotating wheel.
5. 20× SSPE solution: 200 mM NaH 2 PO 4 , pH 7.4, 3.6 M NaCl, 20 mM EDTA (5).
6. 20× SSC: 300 mM sodium citrate, pH 7.0, 3 M NaCl (5).
7. 50× Denhardt’s solution: 1% each of BSA, Ficoll 400, and PVP (5).
8. 20% SDS.
9. Sonicated salmon sperm DNA.