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

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4. 100% and 70% ethanol.
5. LoTE: 3 mM Tris-HCl (pH 7.5), 0.2 mM EDTA (pH 7.5).
6. TE: 10 mM Tris-HCl (pH 7.5), 1 mM EDTA (pH 7.5).
7. (Optional) QIAquick ® DNA Cleanup System: QIAquick Nucleotide Removal and PCR
Purification Kits; and, for higher through-put, QIAquick 8 PCR Purification Kit with
QIAvac 6S (Qiagen Ltd, West Sussex, UK).
2.4. Oligonucleotides
1. Biotinylated oligo dT: 5′ [biotin] T 18 (Alternative protocols, see Note 3).
2. SAGE Linker 1A: 5′ TTT GGA TTT GCT GGT GCA GTA CAA CTA GGC TTA ATA
GGG ACA TG 3′
3. SAGE Linker 1B: 5′ TCC CTA TTA AGC CTA GTT GTA CTG CAC CAG CAA ATC C
[amino mod. C7] 3′
4. SAGE Linker 2A: 5′ TTT CTG CTC GAA TTC AAG CTT CTA ACG ATG TAC GGG
GAC ATG 3′
5. SAGE Linker 2B: 5′ TCC CCG TAC ATC GTT AGA AGC TTG AAT TCG AGC AG
[amino mod. C7] 3′
6. SAGE Primer 1: 5′ [biotin] GGA TTT GCT GGT GCA GTA CA 3′ (11)
7. SAGE Primer 2: 5′ [biotin] CTG CTC GAA TTC AAG CTT CT 3′ (11)
8. M13 Forward: 5′ GTA AAA CGA CGG CCA GT 3′
9. M13 Reverse: 5′ GGA AAC AGC TAT GAC CAT G 3′
The working concentration of all primers, except biotinylated oligo dT, is 350 ng/µL.
Obtain linkers and biotinylated oligos gel-purified: recommended suppliers include
Integrated DNA Technologies, Inc. (IA, USA) and Oswel Research Products Ltd.
(Southampton, UK). Before use, linkers must be kinased, either biochemically during
synthesis or later enzymatically (see Note 4), and annealed to the complementary
linker.
2.5. Restriction and Modifying Enzymes
1. NlaIII (10 U/µL), supplied with NEBuffer 4 and 100× bovine serum albumin (New
England Biolabs [NEB] Inc, MA,) stored at –70°C).
2. BsmFI (2 U/µL) (NEB).
3. SphI (5 U/µL) (NEB).
4. T4 Polynucleotide Kinase 10U/µL (NEB) (or obtain linker oligonucleotides already
kinased, see Note 4).
5. T4 DNA Ligase (5 U/µL, or 20 U/µL if preferred; NEB). Aliquot ligase enzyme and buffer
to prevent cross-contamination from PCR products.
6. DNA polymerase I large fragment (Klenow); 5 U/µL (NEB).
2.6. Magnetic Beads and Related Materials
1. Dynabeads ® M-280 Streptavidin (-coated) (Dynal AS, Oslo, Norway).
2. Magnetic particle concentrator (magnetic stand to immobilize beads) and sample mixer
(Dynal AS).
3. 2× Binding and washing (B+W) buffer: 2 M NaCl, 10 mM Tris-HCl (pH 7.5), 1 mM
EDTA. Also prepare 1× B+W buffer solution.
4. (Optional) Nonstick RNase-free tubes (Ambion) have been recommended for use with
magnetic beads to minimize adhesion of beads to the tube walls.
5. (Alternative protocols: Dynabeads ® Oligo dT 25 (Dynal AS; see Note 3 and Subheadings
2.2. and 2.4.).