DNA/RNA preparation

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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)