epitope tags for detection and purification(e.g, His tag). When PCR is involved, the gene will eventually need to be entirely re sequenced in order to rule out PCR-induced mutations that can occur at a low frequency. If mutations are found, they will need to be repaired, thereby adding to the time required to generate the final expression construct. The best practice is to start with a high-fidelity polymerase with a proofreading (3-5 exonuclease activity) function to avoid PCR errors. Sequence information If you are lucky enough to obtain a dnA from a known source a new litany of questions will need to be answered Is a sequence and restriction map available? Do you know what vector the gene has been cloned into? Has the gene been sequenced in its entirety? How much do you trust the source from which you have received the gene? It is usually best to have the gene re-sequenced so that you know the junctions and restriction sites and can assure yourself that you are indeed working with the correct gene. What do you do if there are differences between your sequence and the published sequence? You will need to decide if the difference is due to a mutation, an artifact from the PCr reaction, a gene poly morphism, or an error in the published sequence. A search of an eSt database coupled with a comparison with genes of other species can help distinguish whether the error is in the data- base or due to a polymorphism. Alternatively, sequencing multi ple, independently derived clones can help answer the ese questions. Control Regions We now have a gene with a confirmed sequence. But which control regions are present? Does the gene contain a Kozak sequence, 5-GCCA/GCCAUGG-3, required to promote effi cient translational initiation of the open reading frame (ORF) in a vertebrate host(Kozak, 1987) or an equivalent sequence 5-CAAAACAUG-3 for expression in an insect host( Cavener, 1987)? If this sequence is missing, it is essential to add it to your expression vector. It is also advisable to trim the gene to remove ny unnecessary sequences upstream of the ATG. The 5non coding regions may contain sequences(e.g, upstream ATG's or secondary structures) that may inhibit translation from the actual start. A noncoding sequence at the 3 end may destabilize the message Eukaryotic Expressionepitope tags for detection and purification (e.g., His6 tag). When PCR is involved, the gene will eventually need to be entirely re￾sequenced in order to rule out PCR-induced mutations that can occur at a low frequency. If mutations are found, they will need to be repaired, thereby adding to the time required to generate the final expression construct. The best practice is to start with a high-fidelity polymerase with a proofreading (3¢–5¢ exonuclease activity) function to avoid PCR errors. Sequence Information If you are lucky enough to obtain a DNA from a known source, a new litany of questions will need to be answered. Is a sequence and restriction map available? Do you know what vector the gene has been cloned into? Has the gene been sequenced in its entirety? How much do you trust the source from which you have received the gene? It is usually best to have the gene re-sequenced so that you know the junctions and restriction sites and can assure yourself that you are indeed working with the correct gene. What do you do if there are differences between your sequence and the published sequence? You will need to decide if the difference is due to a mutation, an artifact from the PCR reaction, a gene poly￾morphism, or an error in the published sequence. A search of an EST database coupled with a comparison with genes of other species can help distinguish whether the error is in the data￾base or due to a polymorphism. Alternatively, sequencing multi￾ple, independently derived clones can also help answer these questions. Control Regions We now have a gene with a confirmed sequence. But which control regions are present? Does the gene contain a Kozak sequence, 5¢-GCCA/GCCAUGG-3¢, required to promote effi- cient translational initiation of the open reading frame (ORF) in a vertebrate host (Kozak, 1987) or an equivalent sequence 5¢-CAAAACAUG-3¢ for expression in an insect host (Cavener, 1987)? If this sequence is missing, it is essential to add it to your expression vector. It is also advisable to trim the gene to remove any unnecessary sequences upstream of the ATG. The 5¢ non￾coding regions may contain sequences (e.g., upstream ATG’s or secondary structures) that may inhibit translation from the actual start. A noncoding sequence at the 3¢ end may destabilize the message. Eukaryotic Expression 499