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Q5 hot start 2 master mix reagent

Manufactured by New England Biolabs

The Q5 Hot Start 2× Master Mix is a high-performance, ready-to-use PCR reagent that contains a chemically modified hot-start DNA polymerase, reaction buffer, dNTPs, and stabilizers. The reagent is designed to provide robust and reliable amplification results.

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Lab products found in correlation

4 protocols using q5 hot start 2 master mix reagent

1

Amplification of Novel MAD-Series Enzymes

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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:

TABLE 1
STEPTEMPERATURETIME
DENATURATION98° C. 30 SEC
30 CYCLES98° C. 10 SEC
66° C. 30 SEC
72° C.2.5 MIN
FINAL72° C.  2 MIN
EXTENSION
HOLD12° C.

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2

Amplification of MADzyme Coding Sequences

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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:

STEPTEMPERATURETIME
DENATURATION98° C.30 SEC
30 CYCLES98° C.10 SEC
66° C.30 SEC
72° C. 3 MIN
FINAL EXTENSION72° C. 2 MIN
HOLD12° C.

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3

Cloning of Novel MAD-Series Enzymes

Check if the same lab product or an alternative is used in the 5 most similar protocols

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:

TABLE 1
STEPTEMPERATURETIME
DENATURATION98° C. 30 SEC
30 CYCLES98° C. 10 SEC
66° C. 30 SEC
72° C.2.5 MIN
FINAL72° C.  2 MIN
EXTENSION
HOLD12° C.

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4

Cloning and Amplification of Novel MAD-Series Enzymes

Check if the same lab product or an alternative is used in the 5 most similar protocols

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:

TABLE 1
STEPTEMPERATURETIME
DENATURATION98° C.30 SEC
30 CYCLES98° C.10 SEC
66° C.30 SEC
72° C.2.5 MIN
FINAL72° C.2 MIN
EXTENSION
HOLD12° C.

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