DNA from all samples was extracted using the cetyl trimethyl ammonium bromide (CTAB) method.50 To obtain 16S V3 and V4 regions, target genes were amplified, and using a specific barcode primer set (341F, 806R).51 (link) All PCR reactions were performed in a 30 μL reaction mixture (15 μL Phusion Master Mix 2X, New England Biolabs; 3 μL [6 μM] primer [2 μM]; 10 μL [5-10 ng] DNA [1 ng/μL]; 2 μL dd H2O). Thermal cycling consisted of an initial denaturation at 98°C for 1 minute, followed by 30 cycles of denaturation at 98°C for 10 seconds, annealing at 50°C for 30 seconds, and extension at 72°C for 30 seconds; a final extension was performed at the end of the run at 72°C for 5 minutes. An identical volume of 1X loading buffer was mixed with the PCR products; then, electrophoresis was performed on a 2% agarose gel for PCR product detection. Bright bands (400-450 bp) were chosen for further experiments. PCR products were mixed at equidensity ratios, and were then extracted with the GeneJET Gel Extraction Kit (Thermo Scientific).
Phusion 2x master mix
Manufactured by New England Biolabs
Phusion 2X Master Mix is a high-fidelity DNA polymerase blend designed for PCR amplification. It provides accurate DNA synthesis with increased processivity and fast extension rates.
Automatically generated - may contain errors
Lab products found in correlation
Genejet gel extraction kit, by Thermo Fisher Scientific (2 mentions)
Dreamtaq green pcr master mix, by Thermo Fisher Scientific (1 mentions)
T4 ligase, by Promega (1 mentions)
Indexed primers, by Illumina (1 mentions)
Td buffer, by Illumina (1 mentions)
Tn5 enzyme, by Illumina (1 mentions)
Minelute kit, by Qiagen (1 mentions)
Quick dna miniprep kit, by Zymo Research (1 mentions)
Q5 2x master mix, by New England Biolabs (1 mentions)
In fusion hd cloning plus kit, by Takara Bio (1 mentions)
T4 polynucleotide kinase, by New England Biolabs (1 mentions)
Wizard genomic dna purification kit, by Promega (1 mentions)
Oligonucleotide primers, by Integrated DNA Technologies (1 mentions)
6 protocols using phusion 2x master mix
1
16S V3-V4 Amplification and Sequencing
2
16S rRNA Gene Amplification and Sequencing
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DNA from all samples was extracted using the CTAB (cetyltrimethylammonium bromide) method. To obtain 16S V3 and V4 regions, target genes were amplified and using a specific barcode primer set (341F, 806R) (Berg et al., 2012; (link)Michelsen et al., 2014) (link). All PCR reactions were performed in a 30 μL reaction mixture (15 μL Phusion Master Mix 2X, New England Biolabs; 3 μL (6 μM) Primer (2 μM); 10 μL (5 -10 ng) DNA (1 ng/μL); 2 μL dd H 2 O). Thermal cycling consisted of initial denaturation at 98°C for 1 min, followed by 30 cycles of denaturation at 98°C for 10 s, annealing at 50°C for 30 s, and extension at 72°C for 30 s, a final extension was performed at the end of the run at 72°C for 5min. We mixed an identical volume of 1X loading buffer with the PCR products, then performed electrophoresis on 2% agarose gel for PCR product detection. We chose 400-450 bp bright bands for further experiments. PCR products were mixed in equidensity ratios, then extracted with GeneJET Gel Extraction Kit (Thermo Scientific).
3
Phusion Master Mix PCR Protocol
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All PCR reactions were performed using a Phusion
2X Master Mix (New England Biolabs) or DreamTaq Green PCR Master Mix
(Thermo Fisher Scientific). T4 ligase (Promega) was used for the DNA
ligation reactions. Restriction enzymes were purchased from Thermo
Fisher Scientific.
2X Master Mix (New England Biolabs) or DreamTaq Green PCR Master Mix
(Thermo Fisher Scientific). T4 ligase (Promega) was used for the DNA
ligation reactions. Restriction enzymes were purchased from Thermo
Fisher Scientific.
4
ATAC-Seq Protocol for Early Embryos
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Early nc14 embryos were placed in ATAC-seq lysis buffer (Buenrostro et al., 2013 (link)) without detergent, with 5% glycerol added. Embryos were then taken out of the freezing solution and placed onto a glass slide which was then put on dry ice for 2 min. Once embryos were completely frozen, the glass slide was removed and embryos were sliced with a razor blade chilled in dry ice. Once sliced embryo halves were moved to tubes containing ATAC-seq lysis buffer with 0.15 mM spermine added to help stabilize chromatin. Embryo halves were then homogenized using single use plastic pestles. IGEPal CA-630 was added to a final concentration of 0.1%. After a 10 min incubation nuclei were spun down and resuspended in water. Twenty halves were added to the transposition reaction containing 25 µl of 2x TD buffer (Illumina), and 2.5 ul of Tn5 enzyme (Illumina) and the reaction was incubated at 37°C for 30 min as in Buenrostro et al. (2013) (link). Transposed DNA was purified using Qiagen Minelute kit. Libraries were then amplified using phusion 2x master mix (NEB) and indexed primers from Illumina. Libraries were then purified with Ampure Beads and sequenced on the Hiseq4000 using 100 bp paired end reads.
5
Genomic Region Amplification and Extraction
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Primers were designed to amplify a genomic region spanning B5X54_RS07480–B5X54_RS07510; a primer was designed to anneal to the B5X54_RS07480 coding region (pyr fwd screen, Additional file 5 : Table S1), and 3′ to the B5X54_RS07510 coding DNA sequence (pyr rev screen, Additional file 5 : Table S1). Genomic DNA was extracted from the respective strains using a Zymo Research Quick-DNA Miniprep Kit (Catalog # D3006, Zymo Research Corp., Irvine, CA) according to manufacturer’s instructions, and used as template in PCR amplification carried out using Phusion 2X Master Mix according to manufacturer’s instructions (Catalog # M0531S, New England Biolabs, Ipswich, MA) (Additional file 6 : Table S3).
6
Fluorescent Lipid II Assay for Pneumococcal Cell Wall Synthesis
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Unless otherwise indicated, all chemicals and reagents were purchased from Sigma-Aldrich. Restriction enzymes, KOD DNA polymerase, Q5 2X Master Mix, Phusion 2X Master Mix, and T4 polynucleotide kinase were purchased from New England Biolabs. The In-fusion HD Cloning Plus kit was purchased from Takara Bio USA. Oligonucleotide primers were purchased from Integrated DNA Technologies (IDT). Culture media were purchased from Becton Dickinson. Streptococcus pneumoniae ΔmurMN Lipid II was isolated from cells as described previously (53 (link), 54 (link)). Lipid II was labeled with ATTO488 as previously described (11 (link)). S. aureus SgtBY181D was expressed and purified as previously reported (55 (link)). Genomic DNA was isolated using a Wizard Genomic DNA Purification kit (Promega).
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