Rtaq dna polymerase

The Fhb7
random mutagenesis library was constructed using error-prone PCR and
MEGAWHOP PCR as described.^{61 (link),62 (link)} Briefly, the fragments
of Fhb7 containing random mutations were obtained by error-prone PCR
with primers Fhb7-F and Fhb7-R using rTaq DNA polymerase (Vazyme Biotech).
The reaction mixture contained 0.2 mM each of dGTP and dATP, 1 mM
each of dTTP and dCTP, 75 μM MnCl₂, 5.5 mM MgCl₂, 60 ng plasmid pQlinkHx-Fhb7 as template. Then, about 500
ng of the fragments were used as a megaprimer and 50 ng plasmid pTrc99a-CysG^A-Fhb7 as a template to perform MEGAWHOP PCR with KOD-Plus-Neo
polymerase (TOYOBO). DpnI was added to the MEGAWHOP PCR products to
digest the wild-type plasmid at 37 °C for 5 h and then inactivation
at 80 °C for 20 min. The digestion products were transformed
into E. coli MC1061 with electro-transformation.
About 10⁴ transformants were recovered. All the transformants
were used for stability screening. Ten randomly picked clones from
the library were sequenced and contained an average of 1–2
amino acid mutations per clone.

Primer and gene synthesis was conducted at
Generay Biotech Co. Ltd. DNA sequencing was performed at RuiBiotech
Company. ClonExpress II One Step Cloning Kit was purchased from Vazyme
Biotech. PCR to obtain DNA fragments was carried out using standard
thermos cycling protocols with TransStart FastPfu DNA polymerase (Transgene
Biotech) and MEGAWHOP PCR was conducted using KOD-Plus-Neo polymerase
purchased from TOYOBO. Error-prone PCR was performed using rTaq DNA
polymerase (Vazyme Biotech). All protein quantification was performed
using a NanoDrop 2000 Spectrophotometer (Thermo). The analysis of
enzymatic reaction products was performed on an ACQUITY ultraperformance
liquid chromatography (UPLC) H-class system (Waters) coupled with
an SQ Detector 2 equipped with an electrospray ionization source.
All the UPLC/mass spectrometry (MS) analysis was conducted on an ACQUITY
UPLC BEN C18 column (length, 50 mm; inner diameter, 2.1 mm; particle
size, 1.7 μm; Waters) at a flow rate of 0.3 mL per minute at
40 °C. The gradient elution consists of 0.1% formic acid (A)
and MeCN (B). The gradient programs were 100% A, 0–0.5 min,
100–98% A, 0.5–1 min, 98–95% A, 1–2.5
min, 95–60% A, 2.5- 4.5 min, 60–10% A, 4.5–6.5
min, 10% A, 6.5–8.0 min.