Q5 hot start 2 master mix reagent

Example 2

The novel MAD-series enzyme coding sequences were cloned into a pUC57 vector with T7-promoter sequence attached to the 5′-end of the coding sequence and a T7-terminator sequence attached to the 3′-end of the coding sequence.

First, Q5 Hot Start 2× master mix reagent (NEB, Ipswich, Mass.) was used to amplify the novel MAD-series sequences using the pUC57 plasmid as a source of MAD-series templates. The forward primer 5′-TTGGGTAACGCCAGGGTTTT [SEQ ID No. 27] and reverse primer 5′-TGTGTGGAATTGTGAGCGGA [SEQ ID No. 28] amplified the sequences flanking the novel MAD-series variant in the pUC57 vector including the T7-promoter and T7-terminator components attached to the MAD7 variant sequence at the 5′- and 3′-end of the novel MAD-series variants, respectively. 1 μM primers and 5 ng/uL pUC57 template were used in PCR reactions to generate linear dsDNA product encoding the novel MAD-series variant. The PCR conditions shown in Table 1 were used:

Example 3

The MADzyme coding sequences were cloned into a pUC57 vector with T7-promoter sequence attached to the 5′-end of the coding sequence and a T7-terminator sequence attached to the 3′-end of the coding sequence.

First, Q5 Hot Start 2× master mix reagent (NEB, Ipswich, Mass.) was used to amplify the MADzyme sequences cloned in the pUC57 vector. The forward primer 5′-TTGGGTAACGCCAGGGTTTT [SEQ ID No. 172] and reverse primer 5′-TGTGTGGAATTGTGAGCGGA [SEQ ID No. 173] amplified the sequences flanking the MADzyme in the pUC57 vector including the T7-promoter and T7-terminator components at the 5′- and 3′-end of the MADzymes, respectively. 1 μM primers were used in a 10 μL PCR reaction using 3.3 μL boiled cell samples as templates in 96 well PCR plates. The PCR conditions shown in Table 2 were used:

Example 2

The novel MAD-series enzyme coding sequences were cloned into a pUC57 vector with T7-promoter sequence attached to the 5′-end of the coding sequence and a T7-terminator sequence attached to the 3′-end of the coding sequence.

First, Q5 Hot Start 2× master mix reagent (NEB, Ipswich, Mass.) was used to amplify the novel MAD-series sequences using the pUC57 plasmid as a source of MAD-series templates. The forward primer 5′-TTGGGTAACGCCAGGGTTTT [SEQ ID No. 27] and reverse primer 5′-TGTGTGGAATTGTGAGCGGA [SEQ ID No. 28] amplified the sequences flanking the novel MAD-series variant in the pUC57 vector including the T7-promoter and T7-terminator components attached to the MAD7 variant sequence at the 5′- and 3′-end of the novel MAD-series variants, respectively. 1 μM primers and 5 ng/uL pUC57 template were used in PCR reactions to generate linear dsDNA product encoding the novel MAD-series variant. The PCR conditions shown in Table 1 were used: