2 Enzymatic Modification of DNA and RNA
47
Subprotocol 8
DNA - Dependent RNA Polymerases
In addition to the DNA polymerases mentioned above, several RNA polymerases are available. They are encoded by bacteriophage (SP6, T7, and T3}
and synthesize RNA on double stranded DNA templates carrying the appropriate bacteriophage-specific promotor. The enzymes are used in vitro
to generatelarge quantities ofRNA complementaryto one strand offoreign
DNA which has been cloned immediately downstream from the promotor
in appropriate plasmids. The RNA can be used for in vitro translation experiments, or, if a radioactive label is incorporated as probe in Northern
blots or in situ hybridizations.
Protocol for in vitro transcription {T7, SP6 RNA polymerase)
RNA polymerases recognize the respective bacteriophage promotor sequence with high specificity. Plasmid vectors (e.g. the pGEM series, Promega) containing a multiple cloning site (MCS) with adjacent T7 or SP6 promotor sequences can be used for the transcription from either strand of a
double-stranded DNA sequence cloned into the MCS. The protocol allows
radiolabeling of RNA to high specific activity for in situ hybridization.
Materials
• Heating block (37°C}
• Plasmid vector containing a T7 or SP6 promotor upstream (i. e. 5'} of the
DNA strandtobe transcribed into RNA. The template must be linearized
by digestion with a restriction enzyme cutting downstream (i. e. 3'} of the
transcribed strand. Avoid restriction enzymes that create protruding 3'
termini. Protruding 3' ends may aberrantly prime transcription of long
RNA molecules. If necessary, refer to the protocol for removal of protruding 3' ends, subprotocol2.6. Plasmid concentration is 0.1 pmol/jll,
i.e. 200 ng/jll for a 3 kb plasmid.
• lOx buffer: 400 mM Tris-HCl pH 7.5, 60 mM MgClz, 20 mM spermidine
HCl, 50 mM NaCl. The buffer should be stored insmall aliquots at -20°C.
• rNTP mix: 5 mM each rATP, rCTP, rGTP
•
[ 35 S]rUTP
Supplies
Reagents
and solutions
47
Subprotocol 8
DNA - Dependent RNA Polymerases
In addition to the DNA polymerases mentioned above, several RNA polymerases are available. They are encoded by bacteriophage (SP6, T7, and T3}
and synthesize RNA on double stranded DNA templates carrying the appropriate bacteriophage-specific promotor. The enzymes are used in vitro
to generatelarge quantities ofRNA complementaryto one strand offoreign
DNA which has been cloned immediately downstream from the promotor
in appropriate plasmids. The RNA can be used for in vitro translation experiments, or, if a radioactive label is incorporated as probe in Northern
blots or in situ hybridizations.
Protocol for in vitro transcription {T7, SP6 RNA polymerase)
RNA polymerases recognize the respective bacteriophage promotor sequence with high specificity. Plasmid vectors (e.g. the pGEM series, Promega) containing a multiple cloning site (MCS) with adjacent T7 or SP6 promotor sequences can be used for the transcription from either strand of a
double-stranded DNA sequence cloned into the MCS. The protocol allows
radiolabeling of RNA to high specific activity for in situ hybridization.
Materials
• Heating block (37°C}
• Plasmid vector containing a T7 or SP6 promotor upstream (i. e. 5'} of the
DNA strandtobe transcribed into RNA. The template must be linearized
by digestion with a restriction enzyme cutting downstream (i. e. 3'} of the
transcribed strand. Avoid restriction enzymes that create protruding 3'
termini. Protruding 3' ends may aberrantly prime transcription of long
RNA molecules. If necessary, refer to the protocol for removal of protruding 3' ends, subprotocol2.6. Plasmid concentration is 0.1 pmol/jll,
i.e. 200 ng/jll for a 3 kb plasmid.
• lOx buffer: 400 mM Tris-HCl pH 7.5, 60 mM MgClz, 20 mM spermidine
HCl, 50 mM NaCl. The buffer should be stored insmall aliquots at -20°C.
• rNTP mix: 5 mM each rATP, rCTP, rGTP
•
[ 35 S]rUTP
Supplies
Reagents
and solutions