1. Template cover slip. Template cover slips should be used for subsequent microdissection.
2. 20X SSC: Adjust the pH to 5.3 with concentrated HCl.
3. 0% Formamide, 2X SSC: Dilute 70 mL of formamide and 10 mL of 20X SSC, pH 5.3, with 20 mL of H2O for a 100-mL total volume. Seal container with parafilm and store at 4°C until ready for use. Heat to 70°C in a Coplin jar for each experiment. Make fresh for each experiment.
4. Ethanol 70%, 85%, and 100% room temperature: Dilute 200 proof ethanol with distilled (d)H2O to 70%, 85%, 50 mL per percent. Store stock at room temperature in a flammable cabinet when not in use.
5. phi29 DNA Polymerase, 10,000 U/mL (NEB, Beverly, MA). Aliquot upon arrival and store at -20°C.
6. 10X phi29 DNA Polymerase buffer: 10X buffer is provided with phi29 DNA Polymerase. Aliquot and store at -20°C upon arrival. 1X buffer is prepared fresh for each experiment by diluting 10 pL of 10X buffer and 2 pL of 10 mg/mL bovine serum albumin in 88 pL of dH2O (see Note 3).
7. dNTP mix: (Roche): Dilute each dNTP (dATP, dCTP, dGTP, dTTP, each at a stock concentration of 100 mM each) in dH2O to a working concentration of 2 mMper dNTP. Aliquot and store at -20°C.
8. 4 mM dithiothreitol (DTT): the DTT used for our experiments was obtained from the 1 M stock vial provided with the Molecular Staging Repli-g kit and diluted with sterile dH2O to 4mM However, this DTT was used by default. DTT purchased from a vendor will be more than adequate.
9. 2 mMRandom hexamers, 5'-nnnnnn-3' where n = A, T, C, or G (Genosys, Woodlands, TX). Primers will arrive dry and will need to be resuspended to 2 mMwith 1X Tris-EDTA buffer, pH 8.0 (Invitrogen, Carlsbad, CA).
10. Clean 24 x 60-mm cover slips.
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