DNA/RNA preparation

In this section:

10x mRNA Transcription Buffer

2x mRNA Nucleotide Triphosphate Mix

TE

Homoginization Buffer

Reticulocyte Standard Buffer (RSB)

Nuclei Freezing Buffer

10x mRNA Transcription Buffer

120 mM MgCl2

800 mM HEPES-Cl (pH 7.5)

20 mM spermidine-HCl

2x mRNA Nucleotide Triphosphate Mix

6 mM ATP

6 mM CTP

6 mM UTP

6 mM GTP

3 mM GTP

9 mM m7(5')Gppp(5')G, cap analog (e.g. New England Biolabs 1404 or Ambion 8050)

• Alternatively, use unmethylated GpppG (e.g. New England Biolabs 1407), which is less expensive. It is reported not to be as good as the methylated analog in reticulocyte translation, but in oocytes and embryos, this reaction yields RNA that has a potency similar to that of Ambion's mMessage Machine (Ambion).

TE

10 mM Tris (pH 7.5)

1 mM EDTA

Homoginization Buffer

1% SDS

10 mM EDTA

20 mM Tris (pH 7.5)

100 mM NaCl

Reticulocyte Standard Buffer (RSB)

10 mM NaCl

10 mM Tris-HCl (ph 8)

5 mM MgCl2

Nuclei Freezing Buffer

50% glycerol

0.15 M NaCl

5 mM MgCl2

10 mM Tris-HCl (pH 8)

0.1 mM EDTA

1 mM DTT (dithiothreitol)