This assay was conducted as reported previously (62 (link)). The FAM-labeled probe covering the promoter region of yfjY was amplified with primer pair FAM-yfjY-P-F and yfjY-P-R, and the products were purified with QIAEX II Gel Extraction Kit (Qiagen, Hilden, Germany). The labeled probes (200 ng) were mixed with varying amounts of MqsA, and the mixtures were incubated for 30 min at 25°C. An orthogonal combination of DNase I (NEB, M0303S) and incubation time were used to achieve the best cutting efficiency. A final concentration of 200 mM EDTA was added to the reaction mixture to stop the reaction. The DNA was purified again with a QIAEX II Gel Extraction Kit (Qiagen, Hilden, Germany), and the generated products were screened and analyzed as reported (62 (link)).
Qiaex 2 gel extraction kit
Manufactured by Qiagen
Sourced in Germany, United States, Netherlands, China, United Kingdom, Italy, Japan
The QIAEX II Gel Extraction Kit is a laboratory equipment product designed for the purification of DNA fragments from agarose or polyacrylamide gels. It allows for the efficient recovery of DNA fragments from gel slices for further downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Pgem t easy vector, by Promega (14 mentions)
Pgem t vector, by Promega (7 mentions)
Qiaprep spin miniprep kit, by Qiagen (7 mentions)
Lightcycler rna master sybr green 1, by Roche (5 mentions)
Genejet plasmid miniprep kit, by Thermo Fisher Scientific (5 mentions)
Bigdye terminator cycle sequencing kit, by Thermo Fisher Scientific (5 mentions)
T4 dna ligase, by New England Biolabs (5 mentions)
Qiaamp dna mini kit, by Qiagen (4 mentions)
Abi prism 3730 dna sequencer, by Thermo Fisher Scientific (4 mentions)
Miseq platform, by Illumina (4 mentions)
Pmd18 t vector, by Takara Bio (4 mentions)
Topo ta cloning kit, by Thermo Fisher Scientific (4 mentions)
Pgem t easy, by Promega (4 mentions)
Dneasy blood tissue kit, by Qiagen (4 mentions)
Lightcycler 1, by Roche (4 mentions)
Dneasy blood and tissue kit, by Qiagen (4 mentions)
Gotaq hot start colorless master mix, by Promega (3 mentions)
Kapa pure beads, by Roche (3 mentions)
Kapa hyper prep kit, by Roche (3 mentions)
Ez dna methylation direct kit, by Zymo Research (3 mentions)
237 protocols using qiaex 2 gel extraction kit
1
MqsA Binding Assay for yfjY Promoter
2
Synthetic Oligonucleotide Purification and Toxin Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All ssDNA oligonucleotides were synthesized and purified by high-performance liquid chromatography (HPLC) by Sangon Biotechnology Co., Ltd. (Shanghai, China). Gymnodimine-A (GYM-A), brevetoxin (BTX), spirolide (SPX), and pinnatoxin (PnTX) were purchased from the National Research Council Canada (Halifax, NS, Canada). Okadaic acid (OA), dinophysistoxin (DTX), palytoxin (PLTX), gonyautoxin (GTX), saxitoxin (STX), microcystin-LR (MC-LR), and nodularin-R (NOD-R) were purchased from Taiwan Algal Science Inc. (Taiwan, China). DynabeadsTM M-270 streptavidin (diameter 2.8 µm) and the Qubit® ssDNA assay kits were purchased from Thermo Fisher Scientic (Chelmsford, MA, USA). GoTaqHot® Start Colorless Master Mix was purchased from Promega Corporation (Fitchburg, WI, USA). QIAEX® II gel extraction kits were purchased from Qiagen (Frankfurt, Germany). Super streptavidin (SSA) sensor biosensors were purchased from ForteBio (Shanghai, China). Selection Buffer (20 mM pH 7.6 Tris-HCl, 100 mm NaCl, 2 mM MgCl2, 5 mM KCl, and 1 mM CaCl2) was purchased from Tiandz (Beijing, China). Selection Buffer was also used in biolayer interferometry (BLI) experiments. All reagents were of analytical grade and were used without further purification or treatment unless specified. All solutions were prepared with ultrapure water.
3
Binary Vector Cloning of Target Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To clone the target gene fragments in the binary silencing vector pK7GWIWG2(I) (Karimi, Inze & Depicker, 2002 (link)) primers were synthesized to amplify fragments flanked by the recombination sequences attL1 and attL2 (Table S2 ). The amplification reactions contained 10 ng of pGEM-T-cloned DNA, 200 µM of each dNTP, 0.4 µM of each primer and 2 U of Phusion DNA polymerase (New England Biolabs, Ipswich, MA, USA) in a final reaction volume of 50 µL. Temperature cycling for amplification was programmed to start at 98 °C for 2 min, followed by 35 cycles at 98 °C for 10 s, 60 °C for 15 s and 72 °C for 15 s, with a final cycle at 72 °C for 5 min. The amplified products were purified from agarose gels using QIAEX II gel extraction kits (Qiagen, Hilden, Germany), quantified, recombined into pK7GWIWG2(I) using LR clonase (Invitrogen) according to the manufacturer’ s instructions and transformed into TOP10 Escherichia coli cells. The presence of the insert was confirmed by amplification and the direction of insertion was verified by digestion and sequencing. Successful constructs were then transformed by heat shock into Agrobacterium tumefaciens GV3101/MP90 cells and confirmed by PCR.
4
Oligonucleotide Synthesis and Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All ssDNA oligonucleotides were synthesized and purified by high-performance liquid chromatography (HPLC) by Sangon Biotechnology Co., Ltd. (Shanghai, China). DA and KA were purchased from the National Research Council Canada (Halifax, NS, Canada). OA, STX, TTX, NOD-R, dinophysistoxin (DTX), and gonyautoxin (GTX) were purchased from Taiwan Algal Science Inc. (Taiwan, China). DynabeadsTM MyOneTM Streptavidin C1 (diameter 1.0 μm) and the Qubit® ssDNA assay kits were purchased from Thermo Fisher Scientific (Chelmsford, MA, USA). GoTaqHot® Start Colorless Master Mix was purchased from Promega Corporation (Fitchburg, WI, USA). QIAEX® II gel extraction kits were purchased from Qiagen (Frankfurt, Germany). Super streptavidin (SSA) sensor biosensors were purchased from Sartorius (Shanghai, China). Selection Buffer (pH 7.4, 8 g/L NaCl, 2.8975 g/L Na2HPO4, 0.1 g/L MgCl2·6H2O, 0.2 g/L KH2PO4, 0.2 g/L KCl, and 0.1 g/L CaCl2) was purchased from Tiandz (Beijing, China). All reagents were of analytical grade and were used without further purification or treatment unless otherwise specified. All the solutions were prepared with ultrapure water.
5
Validating WGBS Data via DMR Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To validate the WGBS data, we randomly selected six DMRs for BSP [54 (link)]. Genomic DNA samples from all TC, TT, SC, and ST replicates were first bisulfite-converted using EZ DNA Methylation-Gold kits. The bisulfite-converted DNA samples were then PCR amplified as previously described using bisulfite-specific primers (Additional file 1 : Table S4), which were designed using Methyl Primer Express v1.0 (Applied Biosystems, USA). The PCR products were separated using 1% agarose gel electrophoresis. Target DNA fragments were then extracted and gel-purified using QIAEX II Gel Extraction Kits (Qiagen, Germany). The purified DNA was ligated with the pGEM-T Easy vector (Promega, USA) and cloned in DH5α cells (Takara, Japan). For each sample, 10 positive clones were selected and sequenced individually using Sanger sequencing.
6
Recombinant TTFC and FlaB Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A DNA fragment encoding the antigenic TTFC polypeptide was polymerase chain reaction-amplified from pTETtac::ttfC [26 (link)] using two pairs of primers: F-ttfC-N/R-ttfC-N and F-ttfC-C/R-ttfC-C (Table 1 ). Similarly, the entire open reading frame of flaB from V. vulnificus CMCP6 was amplified from pCMM250 [9 (link)] using two primer pairs: F-ttfC-N/R-ttfC-N and F-ttfC-C/R-ttfC-C (Table 1 ).
Amplified DNA fragments were initially cloned into the pCR2.1 TOPO vector (Invitrogen, Inc., Carlsbad, CA, USA), yielding plasmids pCMM8209, pCMM8210, pCMM8211, and pCMM8212. DNA fragments were excised using appropriate restriction enzymes and isolated from agarose gels using the QIAEX II gel extraction kit (Qiagen, Hilden, Germany). Plasmid DNA was purified using a QIAprep Spin Miniprep Kit 250 (Qiagen). The 1.3 kb ttfC and 1.1 kb flaB fragments were then cloned into the pTYB12 vector (New England BioLabs, Beverly, MA, USA), yielding plasmids pCMM8213, pCMM8214, pCMM8215, and pCMM8216 (Table 2 , Fig. 1A ). DNA sequences of the resulting expression vectors were confirmed by the dideoxy-chain termination method. Structure prediction was performed as previously described [28 (link)].
Amplified DNA fragments were initially cloned into the pCR2.1 TOPO vector (Invitrogen, Inc., Carlsbad, CA, USA), yielding plasmids pCMM8209, pCMM8210, pCMM8211, and pCMM8212. DNA fragments were excised using appropriate restriction enzymes and isolated from agarose gels using the QIAEX II gel extraction kit (Qiagen, Hilden, Germany). Plasmid DNA was purified using a QIAprep Spin Miniprep Kit 250 (Qiagen). The 1.3 kb ttfC and 1.1 kb flaB fragments were then cloned into the pTYB12 vector (New England BioLabs, Beverly, MA, USA), yielding plasmids pCMM8213, pCMM8214, pCMM8215, and pCMM8216 (
7
Purification and Analysis of PCR Products
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Example 3
Analysis and Purification of PCR Products and Enzymatic Digestion Products
Purification of desired PCR fragments was carried out using a MinElute PCR Purification Kit (Qiagen, via Westburg, Leusden, The Netherlands; product #28006), according to the manufacturer's instructions. Isolated DNA was quantified by UV spectroscopy and the quality was assessed by agarose gel electrophoresis.
Alternatively, PCR or digestion products were separated by agarose gel electrophoresis (for instance when multiple fragments were present) using a 1% Tris Acetate EDTA agarose gel. The desired fragment was excised from the gel and recovered using the QIAEX II Gel Extraction Kit (Qiagen; product #20051), according to the manufacturer's instructions.
8
Molecular Cloning and Sequencing of LIR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primers viz. PR For and PR Rev were designed on the basis of the submitted nucleotide sequence (KF767352) for amplification of LIR (Table 1 ). The amplification was performed in 50 μl of reaction mixture containing 100 ng of template DNA (RCA product), 50 pmol of each primer, 1x Taq DNA polymerase buffer, 200 μM dNTPs and 1.25 U AmpliTaq DNA Polymerase (Applied Biosystems, USA). The reactions were conducted at 94°C for 5 min as initial denaturation followed by 30 cycles of 94°C for 1 min, 48°C for 2 min, 72°C for 1 min and final extension at 72°C for 5 min. The amplified DNA product was run on 1.5% agarose gel, stained with EtBr and visualized in Gel documentation system (BIO-RAD, USA). The PCR product of the expected size ca. 350 bp was recovered from the agarose gel and purified by using QIAEXII Gel Extraction Kit (QIAGEN, Germany). The quantity and purity of gel eluted DNA were measured by BioPhotometer plus (Eppendorf, Germany) and cloned into pDrive PCR cloning vector (QIAGEN, Germany). The positive colonies were confirmed by PCR and restriction digestion, and nucleotide sequences were determined using an automated sequencer (ABI3730XL, Applied Biosystems, USA).
9
Plasmid Verification and Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The DNA sequences of all plasmids were verified by chain termination sequencing and the plasmids were purified using the QIAfilter Plasmid Midi Kit (for chick electroporation, Qiagen, Hilden, Germany) or EndoFree Plasmid Maxi Kit (for mouse transgenesis, Qiagen). All transgenes for mouse microinjection were excised from ASSinsBBins vectors as SalI- or NotI-fragments (5.7 kb fragment, for transient transgenesis) or a KpnI(5')-SacII(3') fragment (3.7 kb fragment, for mSix1-8-NLSCre line), run on an agarose gel and purified using QIAEX II gel extraction kit (Qiagen). Table 2 lists all plasmids used in this study.
10
Quantitative RT-PCR Analysis of Nitrogen Fixation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Quantitative reverse transcription PCR (qRT-PCR) was conducted using a LightCycler 1.5 instrument with LightCycler RNA Master SYBR Green I (Roche, Basel, Switzerland) following the manufacturer's protocol (Kouzuma et al., 2012 (link)). Briefly, a PCR reaction mixture contained 25-ng total RNA, 1.3 μl of 50-mM Mn(OAc)2 solution, 7.5-μl of LightCycler RNA Master SYBR Green I (Roche), and 0.15 μM primer sets [16S rRNA: 341F-518R (Luo et al., 2010 (link)), nifD: qrt-nifD-F 5′-GGTTGTGCTTATGCAGGATG-3′ and trt-nifD-R 5′-TCCTATAGGTCCGTGTGTGATG-3′, nifK: qrt-nifK-F 5′-GTTGCATTACTTGGAGATCCTG-3′ and qrt-nifK-R 5′-CTTCTGCAAGCATAGCATCG-3′) (Supplementary Figure S1I ]. The DNA fragments of target genes (16S rRNA, nifD, and nifK) were amplified by PCR using Ex Taq polymerase (Takara Bio Inc., Shiga, Japan) and the same primer sets and purified by gel electrophoresis using a QIAEXII gel extraction kit (Qiagen, Venlo, Netherlands) according to the manufacturer's instructions to generate a standard curve. Standard curves were generated by amplifying a series of purified DNA fragments of each gene. Expression levels of target genes (nifD and nifK) were normalized based on the reference gene expression levels (16S rRNA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!