cDNA of the full-length BVF genome was synthesized from total RNA by using Superscript IV reverse transcriptase (Invitrogen) with the primer BVF_NotIEcoRI Rev (Table 1 ). The entire viral genome (6.8 kb) was amplified with the CloneAmp HIFI PCR mix (Takara) using the primers BVF_XbaI Fw and BVF_NotIEcoRI Rev (Table 1 ). The forward primer BVF_XbaIT7 Fw contained an engineered XbaI site and the T7 promoter sequence followed by part of the BVF 5′ end sequence of the viral genome. The reverse primer BVF_NotIEcoRI Rev introduced a poly (A)18 tail and NotI and EcoRI sites at the 3′ end of the viral genome for linearization and cloning purposes. The resultant PCR product was gel purified with NucleoSpin Gel and PCR Clean-up (Takara, San Jose, CA, USA) and ligated into pUC19 vector between XbaI and EcoRI sites by using the T4 DNA ligase (ThermoScientific, Waltham, MA, USA) to generate the clone pUC19-BVF. Recombinant plasmid DNA were extracted and purified using the Zyppy plasmid miniprep kit (Zymo Research, Irvine, CA, USA) and checked for the presence of the BVF genome by restriction digestion and sanger sequencing. The strategy followed to construct the full-length cDNA clone of BVF is shown in Figure 3A.
Cloneamp hifi pcr mix
Manufactured by Takara Bio
CloneAmp HiFi PCR mix is a high-fidelity, ready-to-use PCR reagent designed for consistent and reliable amplification of DNA templates. The mix contains all the necessary components for efficient PCR, including a high-performance DNA polymerase, dNTPs, and buffer.
Automatically generated - may contain errors
Lab products found in correlation
Superscript 4 one step rt pcr system, by Thermo Fisher Scientific (3 mentions)
T4 dna ligase, by Thermo Fisher Scientific (2 mentions)
Nucleospin gel and pcr clean up, by Takara Bio (1 mentions)
Superscript 4, by Thermo Fisher Scientific (1 mentions)
Zyppy plasmid miniprep kit, by Zymo Research (1 mentions)
Sanger sequencing, by Macherey-Nagel (1 mentions)
In fusion hd cloning, by Takara Bio (1 mentions)
Gotaq green master mix, by Promega (1 mentions)
Nucleospin gel and pcr clean up kit, by Macherey-Nagel (1 mentions)
Nucleobond xtra midi kit, by Macherey-Nagel (1 mentions)
In fusion, by Takara Bio (1 mentions)
4 protocols using cloneamp hifi pcr mix
1
Full-Length cDNA Cloning of BVF Genome
2
Constructing Recombinant Bacterial Plasmids
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Bacterial strains and plasmids constructed and used in this study are listed in Table 1 . All cloning work was performed in Escherichia coli DH5α. Vectors (when relevant) and all genes were PCR amplified with CloneAmp HiFi PCR mix (TaKaRa Bio), according to the producer’s instructions. Primers used are listed in Additional file 2 : Table S2. Plasmid pBV2xp was cut with restriction enzymes SacI and BamHI and joined with PCR-amplified sfGFP or α-amylase- and protease (with their native signal peptides) coding genes by the Gibson assembly reaction [97 (link)]. For construction of plasmids harboring α-amylase- and sfGFP-encoding genes with heterologous signal peptides, vectors were PCR amplified as described above and joined with sfGFP- or α-amylase-encoding genes by the Gibson assembly reaction [97 (link)].
3
Influenza A Virus Reverse Genetics
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Two microliters of extracted viral RNA were used as template to amplify the seven genomic segments individually with segment-specific oligonucleotides using the Superscript IV one-step RT-PCR system (Thermo Fisher Scientific), according to manufacturer guidelines. The bidirectional pHW2000-vector, kindly provided by Prof. Dr. Martin Schwemmle, University of Freiburg, Germany, was amplified using CloneAmp HiFi PCR mix (Takara) in a 2-step PCR-program of 98 °C for 10 s followed by 68 °C for 4 min, with 35 cycles. Both PCR products were excised from a 1% agarose gel and extracted using the Nucleospin Gel and PCR clean-up kit (Macherey-Nagel) and used as templates for In-Fusion HD cloning (Takara). Positive constructs were identified by colony PCR using the segment-specific PCR primers and GoTaq green master mix (Promega) with the following cycle profile; 94 °C 5 min, followed by 25 cycles of 94 °C, 1 min, 55 °C 1 min, 72 °C 2 min, and a final elongation step of 7 min at 72 °C. All plasmids were isolated using the Nucleobond Xtra midi kit (Macherey-Nagel) and verified by Sanger sequencing (Microsynth).
4
Cloning and Mutagenesis of IgA1 Proteases
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All inserts of G. haemolysans IgA1P were PCR amplified from chromosomal DNA (Gha_ATCC10379) obtained from the Leibniz Institute DSMZ – German Collection of Microorganisms and Cell Cultures GmbH (Braunschweig, Germany). PCR products coding for G. haemolysans IgA1P residues 907-2201 and 651-2201 were inserted into a pET21b vector encoding for an N-terminal MBP, a thrombin cleavage site, a BamH1 site for PCR product insertion, and a C-terminal 6xHis tag using the single BamH1 site. The G. haemolysans IgA1P E1848A mutation and the Δ 1297-1306 deletion mutant within the construct containing 907-2201 was produced by first PCR amplifying a 3’ PCR fragment using the primers that included the required mutation and then utilizing this PCR fragment as the 3’ primer with the 5’ primer specific for the N-terminal fragment of 907-2201 for a second reaction to produce the full 907-2201 product with the encoded mutation. PCR products for the GhTrp domain that included 651-896 and 684-896 were inserted into pET21b with an N-terminal 6xHis tag and thrombin cleavage site using the single NdeI site. CloneAmp HiFi PCR mix (Takara) was used for all PCR reactions and ligase-independent cloning using In-Fusion (Takara) was used for ligation-independent cloning. All plasmids were confirmed by sequencing. The production of S. pneumoniae IgA1P and its mutant have been previously described9 .
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