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)