Oligonucleotide primers used in this study are listed in Additional file 3 : Table S3. The reaction mixture consisted of 5 μl of 2 × KOD FX neo buffer (Toyobo, Osaka, Japan), 2 μl of 2 mM dNTPs, 0.2 μl of KOD FX neo polymerase (Toyobo), and 0.3 μl each of the primer pair (10 μΜ) in a total volume of 10 μl with sterile water. Cycling conditions were as follows: 94°C for 2 min, followed by 30 cycles each of 98°C for 10 s, 65°C for 30 s, and 68°C for 3–4 min. For construction of pKM152, KOD plus polymerase (Toyobo) was used. This reaction mixture consisted of 1 μl of 10 × KOD plus buffer, 1 μl of 2 mM dNTPs, 0.4 μl of 25 mM MgSO4, 0.2 μl of KOD plus polymerase, and 0.3 μl each of the primer pair (10 μΜ) in a total volume of 10 μl with sterile water. Cycling conditions were as follows: 94°C for 1 min, followed by 30 cycles each of 94°C for 20 s, 60 or 65°C for 30 s, and 68°C for 1–4 min. The amplified DNA fragments were directly used for yeast transformation.
Kod fx neo buffer
Manufactured by Toyobo
Sourced in Japan
KOD FX neo buffer is a specialized buffer solution designed for use with the KOD FX neo DNA polymerase, a high-fidelity enzyme commonly used in PCR amplification. The buffer provides an optimal chemical environment for the enzyme to function effectively, ensuring reliable and accurate DNA replication.
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Lab products found in correlation
3 protocols using kod fx neo buffer
1
Optimized PCR Amplification Conditions
2
Bacterial 16S rRNA gene amplification
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Two universal primers (27F: 5′-AGA GTT TGA TCM TGG CTC AG-3′ and 534R: 5′-ATT ACC GCG GCT GCT GG-3′) [47 (link)] were used to amplify the bacterial 16S rRNA gene-coding region. Amplification reactions were performed in a total volume of 50 μL containing 6 μL of extracted gDNA as the template, 6.5 μL of ddH2O, 25 μL of 2× KOD Fx Neo Buffer, 10 μL of 2 mM deoxyribonucleotide triphosphate, 0.75 μL of 20 mM primers 27F and 534R and 1 μL of KOD Fx Neo polymerase (Toyobo Co., Ltd., Osaka, Japan). PCR amplification was performed using a standard thermocycler (Biometra GmbH, Göttingen, Germany) under the following parameters: 95 °C for 5 min, followed by 30 cycles at 95 °C for 30 s, 63 °C for 30 s, 68 °C for 40 s and a final extension at 68 °C for 6 min. The DNA sequences of the PCR products were analysed using the Basic Local Alignment Search Tool (http://www.ncbi.nlm.nih.gov/BLAST ) for species identification.
3
Bacterial 16S rRNA gene amplification
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Amplification of the V1–V3 regions of 16S rRNA gene was performed using two universal primers: 27F-modification (5′-AGA GTT TGA TC[A/C] TGG CTC AG-3′) and 534R (5′-ATT ACC GCG GCT GCT GG-3′). The polymerase chain reaction (PCR) amplification was achieved in a total volume of 50 μL comprising 6.5 μL of ddH2O, 25 μL of 2× KOD FX Neo buffer (Toyobo, Japan), 10 μL of 2-mM dNTPs, 0.75 μL each of 20-mM primer 27F, and 534R, 6 μL of bacteria DNA template, and 1 μL of KOD FX Neo polymerase (Toyobo, Japan). The amplicons were sequenced using Sanger sequencing by1st Base, Singapore). During PCR-sequencing, we employed g-DNAs of reference bacteria as control.
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