Target Discovery and Validation Reviews and Protocols

64 Beaty et al.
3. The 12% polyacrylamide gel electrophoresis-Tris acetate-EDTA (PAGE-TAE) gel
may be made from the following:
a. 40% Polyacrylamide: 19:1 acrylamide:bis (cat. no. 161-0144, Bio-Rad).
b. 50X TAE buffer (cat. no. 330-008-161, Quality Biological) (see Note 1).
2.2.2. First- and Second-Strand cDNA Synthesis (Step 2)
1. Dynabeads oligo(dT)25 mRNA direct kit (cat. no. 610.11, Dynal).
2. Dynal magnetic particle concentrators (MPC-S cat. no. 120.20, Dynal).
3. Superscript Choice System cDNA synthesis kit (cat. no. 18090-019, Invitrogen).
4. 0.5 M EDTA (cat. no. E7889, Sigma).
5. SDS (cat. no. L4390, Sigma).
2.2.3. Cleavage of cDNA With Anchoring Enzyme (NlaIII)(Step 3)
1. NlaIII (cat. no. 125S, New England Biolabs).
2.2.4. Ligating Linkers top cDNA (Step 4)
1. T4 Ligase (high concentration 5 U/µL; cat. no. 15224-041, Invitrogen).
2. BSA (cat. no. B9001S, New England Biolabs).
2.2.5. Release Tags Using Tagging Enzyme BsmfI of cDNA (Step 5)
1. BmsfI (cat. no. 572S, New England Biolabs).
2. Phenol-chloroform.
3. 0.5 M EDTA (cat. no. P2069, Sigma).
4. LoTE: 3 mM Tris-HCl, pH 7.5, 0.2 mM EDTA, pH 7.5.
2.2.6. Blunt-Ending Release Tags (Step 6)
1. 2 U/L Klenow (cat. no. 27-0929-01, Pharmacia).
2. 7.5 M Ammonium acetate (cat. no. A2706, Sigma).
2.2.7. Ligate to Form Ditags (Step 7)
1. T4 Ligase (high concentration 5 U/µL; cat. no. 15224-041, Invitrogen).
2.2.8. PCR Amplification of Ditags (Step 8)
1. Primer 1: 5′-GGA TTT GCT GGT GCA GTA CA-3′.
2. Primer 2: 5′-CTG CTC GAA TTC AAG CTT CT-3′.
3. 40% Polyacrylamide: 19:1 acrylamide:bis (cat. no. 161-0144, Bio-Rad) for making
12% PAGE-TAE gel.
4. 96-Well plates for PCR (cat. no. Z37,490-3, Sigma).
5. Taq polymerase (cat. no. 10996-034, Invitrogen).
2.2.9. Isolation of Ditags (Step 9)
1. SYBR Green I (cat. no. S-7567, Invitrogen).
2. SpinX filtration microcentrifuge tubes (Costar, Cambridge, MA).