34
KAI-OLAF NETZER
Ribonuclease (RNase A)
RNase Ais isolated from bovine pancreas.lt is an endonuclease specific for
RNA. lt hydrolizes the phosphodiester bound on the 3' side of pyrimidine
residues. RNase works in the absence of cofactors and divalent cations. The
enzyme is used after hybridization experiments to remove unhybridized
RNA probes. Another use is to cleave single base mismatches in DNA:RNA
hybrids in order to map single base mutations. Cantamination with RNase
is often a problern when working with RNA. lts action can be inhibited by
placental RNase inhibitor (RNasinTM' Promega) or by vanadyl-ribonucleoside complexes.
Ligases
Ligasesare isolated from E. coli (E. coli DNA ligase) or from bacteriophage
T4-infected bacteria (T4 DNA ligase).
T4ligase is used more frequently since it is able to join blunt-ended DNA
fragments. lt is a 68 kD enzyme that catalyzes the formation of phosphodiester bonds between adjacent 3' -hydroxyl and 5' -phosphate termini in
DNA (two dephosphorylated fragments cannot be ligated). ATP (contained
in the reaction buffer) supplies energy for the two step reaction. Joining
DNA fragments with cohesive ends is very efficient. Joining DNA fragments
with blunt ends, however, is much slower and requires more enzyme.
Materials
For T 4 ligase reaction
Supplies
• Cooling waterbath {16°C)
• Alternatively: Thermal cycler, programmed to l6°C/hold
Reagents
• DNA, purified and dissolved in waterat approximately O.l!lgl!ll; Vector
DNA should be dephosphorylated prior to ligation in order to prevent
autoligation. The presence of EDT A should be avoided since it may reduce enzyme activity.
• Buffer: lOx T4 DNA ligase buffer (usually supplied bythe manufacturer)
is 0.5 M Tris-HCl, pH7.5, 100 mM MgCh, 100 mM DTT, 10 mM ATP, 250
!lglml bovine serum albumine; for ligation ofblunt ends, use lOx blunt
KAI-OLAF NETZER
Ribonuclease (RNase A)
RNase Ais isolated from bovine pancreas.lt is an endonuclease specific for
RNA. lt hydrolizes the phosphodiester bound on the 3' side of pyrimidine
residues. RNase works in the absence of cofactors and divalent cations. The
enzyme is used after hybridization experiments to remove unhybridized
RNA probes. Another use is to cleave single base mismatches in DNA:RNA
hybrids in order to map single base mutations. Cantamination with RNase
is often a problern when working with RNA. lts action can be inhibited by
placental RNase inhibitor (RNasinTM' Promega) or by vanadyl-ribonucleoside complexes.
Ligases
Ligasesare isolated from E. coli (E. coli DNA ligase) or from bacteriophage
T4-infected bacteria (T4 DNA ligase).
T4ligase is used more frequently since it is able to join blunt-ended DNA
fragments. lt is a 68 kD enzyme that catalyzes the formation of phosphodiester bonds between adjacent 3' -hydroxyl and 5' -phosphate termini in
DNA (two dephosphorylated fragments cannot be ligated). ATP (contained
in the reaction buffer) supplies energy for the two step reaction. Joining
DNA fragments with cohesive ends is very efficient. Joining DNA fragments
with blunt ends, however, is much slower and requires more enzyme.
Materials
For T 4 ligase reaction
Supplies
• Cooling waterbath {16°C)
• Alternatively: Thermal cycler, programmed to l6°C/hold
Reagents
• DNA, purified and dissolved in waterat approximately O.l!lgl!ll; Vector
DNA should be dephosphorylated prior to ligation in order to prevent
autoligation. The presence of EDT A should be avoided since it may reduce enzyme activity.
• Buffer: lOx T4 DNA ligase buffer (usually supplied bythe manufacturer)
is 0.5 M Tris-HCl, pH7.5, 100 mM MgCh, 100 mM DTT, 10 mM ATP, 250
!lglml bovine serum albumine; for ligation ofblunt ends, use lOx blunt