Luria broth and plates were used for the growth of all bacterial strains, supplemented as needed with 25 μg/ml chloramphenicol or 25 μg/ml kanamycin. Phusion High Fidelity DNA polymerase master mix and restriction enzymes were purchased from New England Biolabs (NEB; Ipswich, MA). B-PER® extraction reagents, HisPurTM Cobalt Spin Columns, and Zeba Desalting Columns were obtained from Thermo-Scientific (Pierce Biotechnology; Rockford, IL, USA).
Phusion high fidelity dna polymerase master mix
Manufactured by New England Biolabs
Phusion High Fidelity DNA polymerase master mix is a ready-to-use solution for high-fidelity DNA amplification. It contains Phusion DNA polymerase, dNTPs, and optimized buffer components.
Automatically generated - may contain errors
Lab products found in correlation
T7 dna polymerase, by New England Biolabs (2 mentions)
Stereo discovery v12, by Zeiss (2 mentions)
Dig rna labeling mix, by Roche (2 mentions)
Sigmaspin post reaction clean up columns, by Merck Group (2 mentions)
Clonejet pcr cloning kit, by Thermo Fisher Scientific (2 mentions)
Zymo gel extraction kit, by Zymo Research (2 mentions)
Axiocam mrc, by Zeiss (2 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Zeba desalting column, by Thermo Fisher Scientific (1 mentions)
B per extraction reagent, by Thermo Fisher Scientific (1 mentions)
Pentr directional topo cloning kit, by Thermo Fisher Scientific (1 mentions)
Nucleospin gel and pcr clean up kit, by Takara Bio (1 mentions)
Primestar gxl dna polymerase, by Takara Bio (1 mentions)
Gibson assembly master mix, by New England Biolabs (1 mentions)
Axiocam mrc camera, by Zeiss (1 mentions)
5 protocols using phusion high fidelity dna polymerase master mix
1
Bacterial Strain Growth and Protein Purification
2
Cloning and Riboprobe Synthesis of Zebrafish smarcad1
3
Cloning and Characterization of Zebrafish KCa Channels
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A whole open reading frame was amplified with RT‐PCR using gene‐specific primers for each identified zebrafish KCa channel gene. Primers were designed in ApeE program using default settings. CACC Kozak sequences were added at the 5′d end of forward primer to prepare direct Topo cloning. Total RNAs were exacted from pooled fish embryos composed of about a hundred 1‐3 dpf fish embryos using TRIzol reagent (Thermo Fisher) according to manufactory guidance. After quality checking and quantification by Nanodrop, Reverse transcription was carried out with the SuperScript III First‐Strand Synthesis System (Thermo Fisher) following manual instructions. Phusion High‐Fidelity DNA Polymerase master mix (New England Biolabs) or PrimeSTAR GXL DNA Polymerase (Takara Bio) were chosen for PCR amplification. PCR primers utilized are listed in Table S2 . PCR products were then purified with NucleoSpin Gel and PCR Clean‐up Kit (Takara Bio). Purified PCR products of each gene were cloned into the pENTR‐D‐TOPO or pDONR221 vector and transformed into Top10 Escherichia coli cells using the pENTR Directional TOPO Cloning Kit (Thermo Scientific) according to its manual instruction. Correct clones with the right gene orientation and sequences were verified with Sanger sequencing. All the zebrafish KCa gene clones are available through Addgene (Plasmid #164951‐164964).
4
Phage Assembly via Gibson Cloning
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Primers were designed to obtain long PCR fragments with overlapping joints suitable for Gibson assembly strategies (Table S2 ). Primers for the integrase deletion were created with artificial 40-bp overlaps. Briefly, a 20-bp primer was created around the integrase gene, and a 20-bp flanking region was added to the 5′ end of the primer from the opposing end of the Δint circular junction. Long PCR was performed using the NEB Phusion High-Fidelity DNA polymerase master mix. PCR conditions were as follows: (i) 98°C for 2 min; (ii) 35 cycles with 1 cycle consisting of 98°C for 30 s, melting temperature (Tm) minus two degrees for 30 s, and 72°C for 1 min/kbp; and (iii) 72°C for 10 min. NEB Gibson Assembly master mix was used to ligate phage fragments together. Then, 0.3 pmol of each long PCR product was incubated at 50°C for 15 min for three-fragment phages (41ZΔ, 42argFΔ, and 44GΔ) and 60 min for four or more fragment phages (52SΔ and 64LΔ).
5
Spatial Expression Analysis of zebrafish smarcad1 Genes
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Full‐length coding regions of zebrafish smarcad1 genes were amplified by RT‐PCR using gene‐specific primers designed according to the current DNA sequence in Ensembl. Phusion® High‐Fidelity DNA Polymerase master mix (New England Biolabs) was used for PCR amplification. PCR primers used here are listed in Table S1 . The PCR products were purified using Zymo Gel Extraction Kit (Zymo Research) before they were cloned into the pJet1.2 vector using the CloneJET PCR Cloning Kit (Thermo Scientific). Gene inserts orientation was verified by Sanger sequencing. Riboprobes were synthesized through in vitro transcription using T7 DNA polymerase (New England Biolabs) and DIG RNA Labeling Mix (Roche). Then, the riboprobes were purified by SigmaSpin™ post‐reaction clean‐up columns (Sigma, S5059). Whole‐mount in situ hybridization was carried out according to our previously published method.54 , 55 For histological analysis, post‐hybridization embryos were equilibrated in 15% sucrose, then 30% sucrose in 20% gelatin, after which they were embedded in 20% gelatin for cryosectioning (6–12 μm). Images were acquired using AxioCam MRc camera on Zeiss SteREO Discovery.V12 and Axio Imager 2 compound microscope.
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