Genomic DNA was isolated from the tip of tails of three to four-week-old founder mice using Wizard Genomic DNA purification Kit (Promega), amplified by PCR, and followed by Sanger sequencing (Quintarabio, CA). Mutations were identified by PCR amplifying a 599 bp fragment encompassing the target sequence, followed by Sanger sequencing. Library preparation and WGS was conducted by the Broad Institute (Cambridge, MA) using Illumina HiSeq X, at a coverage of 60X using 150 bp paired-end reads (Supplementary Data 1 ).
Sanger sequencing
Manufactured by Promega
Sourced in United States
Sanger sequencing is a method for determining the nucleotide sequence of DNA. It employs chain-termination DNA sequencing and uses fluorescently labeled dideoxynucleotides to identify the individual nucleotides in a DNA sample.
Automatically generated - may contain errors
Lab products found in correlation
Pgem t easy, by Promega (3 mentions)
Geneart strings, by Thermo Fisher Scientific (2 mentions)
Goscript system, by Promega (2 mentions)
Gotaq polymerase, by Promega (2 mentions)
Hiseq x, by Illumina (1 mentions)
Wizard genomic dna purification kit, by Promega (1 mentions)
Prl tk renilla luciferase reporter vector, by Promega (1 mentions)
Transit lt1 transfection reagent, by Mirus Bio (1 mentions)
T4 rna ligase 1, by New England Biolabs (1 mentions)
Pwo polymerase, by Roche (1 mentions)
Gotaq green, by Promega (1 mentions)
Expin gel sv, by GeneAll (1 mentions)
Pgem t easy vector, by Promega (1 mentions)
T100 thermal cycler, by Bio-Rad (1 mentions)
Custom primers, by Integrated DNA Technologies (1 mentions)
Phusion polymerase, by Integrated DNA Technologies (1 mentions)
Purelink quick plasmid miniprep kit, by Thermo Fisher Scientific (1 mentions)
Exosap it, by Thermo Fisher Scientific (1 mentions)
Zymoclean gel dna recovery kit, by Zymo Research (1 mentions)
Pfn210a ss halotag cmv neo flexi vector, by Promega (1 mentions)
10 protocols using sanger sequencing
1
Genomic DNA Isolation and Characterization
2
Dual luciferase reporter assay for gene regulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genomic regions-of-interest were PCR-amplified from commercial human DNA (Roche, catalog no. 11691112001) using the Q5 kit (New England Bioloabs), cloned into reporter vector pGL4.23 (Promega, catalog no. E841A) and sequence verified by Sanger sequencing (GATC Biotech). About 8000 293FT cells were transfected using the TransIT LT1 transfection reagent (Mirus, catalog no. MIR 2304) with 50 ng plasmid mix consisting of 20 ng of the reporter construct or empty vector pGL4.23, 10 ng of the pRL-TK Renilla luciferase reporter vector (Promega, catalog no. E2241) and, as stuffer DNA, 20 ng of a pGL3 (Promega) deletion derivative with a non-functional luciferase gene. The dual luciferase readout was performed with nearly confluent cells using the SpectraMax M5 (Berthold, Bad Wildbad, Germany); the firefly luciferase reporter signals were normalized to the renilla luciferase transfection control signals. Mean values were calculated from six parallel measurements per transfection (technical replicates), and at least three independent transfections were performed per construct (biological replicates). Statistical evaluation of differences between mean values and empty vector (values of normalized signals set to 1) was performed by one-sample t-test.)
3
Primer Extension and Ligation Technique for cDNA Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Primer extension was performed on RNA from a WT strain as previously described (Diwa et al. 2000 (link)), using a primer with the sequence 5′-CAGAATCAAACGCTTCATAG-3′. The cDNA product was precipitated and 1 µL of the cDNA product was added to 1 µL of a 5′ phosphorylated, 3′ blocked oligonucleotide (5′-AGATCGGAAGAGCACACGTCT-NH2-3′) at 1 µM and 1 µL of dimethyl sulfoxide. The mix was heated to 75°C for three min and placed on ice. The reaction was supplemented with 3 µL of T4 RNA ligase I (NEB), 1.5 µL of 10× reaction buffer, 6.5 µL of 50% PEG-8000, and 1 µL of 10 mM ATP. The reaction was incubated at room temperature for 1 h and an aliquot was used to amplify the ligation product using primers with the sequences 5′-CCTAAGTAATTGAATACTTC-3′ and 5′-AGACGTGTGCTCTTCCGATCT-3′. The PCR product was cloned into pGEM-T easy (Promega), amplified by using primers with the sequences 5′-AGTCACGACGTTGTAAAACG-3′ and 5′-GAGCGGATAACAATTTCACACAGG-3′, and the resulting product was sequenced using Sanger sequencing (Genewiz).
4
Molecular Characterization of Planarian Stem Cell Genes
5
Constructing A. baumannii Mutants and Variants
Check if the same lab product or an alternative is used in the 5 most similar protocols
A. baumannii mutants or variants were constructed using a standard allelic exchange approach with the counterselectable suicide vectors pGP704-Sac28 or pGP704Sac-kan (71 (link), 72 (link)). Derivatives of these plasmids containing deletion constructs of the respective genes or site-directed allele exchanges (of pilA) were transferred to A. baumannii through mating with E. coli S17-1λpir. The deletion constructs were based on the PCR amplification of the flanking regions of the desired genetic regions and, when needed, the aph cassette (Kanr) as the selection marker using PWO polymerase (Roche) or Q5 polymerase (BioLabs). Site-directed changes in the pilA allele were inserted using modified primers. The amplified fragments were joined by overlap extension PCR or Golden Gate assembly (73 (link)) and were cloned inside the suicide plasmids. The correct cloning products were screened for by colony PCR of the E. coli transformants (with GoTaq polymerase; Promega) and ultimately confirmed by Sanger sequencing (Microsynth, Switzerland).
Construction of inducible genes-of-interest was accomplished by placing the respective gene under the control of the arabinose-inducible promoter PBAD inside the miniTn7 transposon TnAraC (23 (link), 63 (link)). The resulting transposons were transferred to the respectiveA. baumannii strains through a standard triparental mating approach (74 (link)).
+ Expand
Construction of inducible genes-of-interest was accomplished by placing the respective gene under the control of the arabinose-inducible promoter PBAD inside the miniTn7 transposon TnAraC (23 (link), 63 (link)). The resulting transposons were transferred to the respective
6
Identifying Bacterial Isolates via Genomic Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For each KPL-named new isolate, Sanger sequencing (Macrogen, USA) was performed using primer 27F on a V1-V3 16S rRNA gene colony-PCR amplicon (GoTaq Green, Promega) of primers 27F and 519R. We assigned each initial isolate to a genus and a putative species based on blastn of each sequence against eHOMDv15.1 (31 ). We then selected a subset of these isolates for whole genome sequencing (WGS). For MSK-named new isolates, all isolates preliminarily assigned to Corynebacterium based on MALDI and/or Sanger sequencing of V1-V3 16S rRNA gene underwent WGS.
7
Molecular Characterization of Planarian Stem Cell Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
PIWI homologs and stem cell progeny markers found in the MA-C2 transcriptome draft were identified using BlastStation Local-64 (TM Software, Arcadia, CA) and corresponding fragments for GdPiwi1 and GdPiwi2 were amplified from G. dorotocephala MA-C2 cDNA. Total RNA extracted as described above was used as a template for reverse transcription using the GoScript™ System (Promega, Madison, WI). Then, the primers TR8465F [5′-GACATGGTGAAGTTCTTGACCTTG-3′] and TR8465R [5′-GTGGATTTTCTACGACAATCACCC-3′] were used to amplify GdPiwi1, whereas TR3618F [5′-GGAGAGACCGATATCCAAGAGTTC-3′] and TR3618R [5′-ATTCCGCATATGACAACGTTCTTC-3′] were used to amplify GdPiwi2. Amplicons were cloned into the pGEM-T Easy (Promega, Madison, WI) and their identity verified through Sanger sequencing (Retrogen Inc., San Diego, CA). Complementary DNA (cDNA) corresponding to GdPROG1-L1, -L2, -L3, GdAGAT-1, GdAGAT-2, and GdCPEB-2 sequences, flanked by SP6 and T3 promoters, were synthesized as GeneArt Strings (ThermoFisher, Waltham, MA). GeneArt construct sequences are provided in Supplementary Table S2 .
8
mtCOI DNA Amplification and Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
mtCOI DNA was amplified using the primer pair HCO-2198 and LCO-1490 [49 (link)]. The PCR process was conducted with a total reaction volume of 20 μL, which included 10 μL SmartGene 2× Dye Mixed Taq (SmartGENE, Daejeon, Republic of Korea), 1 μL of each primer (10 pmol/μL), 5 μL of nuclease-free water and 3 μL of template DNA solution (40 ng). The reaction mixtures underwent amplification with the following parameters: an initial denaturation at 94 °C for 5 min; this was followed by 35 cycles of 94 °C for 30 s, 52 °C for 30 s, and 72 °C for 60 s; and a final extension at 72 °C for 5 min. This process was carried out in a T100 Thermal Cycler from Bio-Rad (Hercules, CA, USA). The PCR products were electrophoresed on a 1% agarose gel, which was then purified using ExpinTM Gel SV (GeneAll, Seoul, Republic of Korea). The PCR amplicons were subcloned into a cloning vector (the pGEM-T Easy vector, Promega, Madison, WI, USA) and sequenced using Sanger sequencing (Macrogen, Seoul, Republic of Korea).
9
Plasmid Construction and Validation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Linear PCR products were produced using Phusion polymerase and custom primers from Integrated DNA Technologies (IDT, Coralville, IA, USA). Templates were E. coli genomic DNA or previously constructed plasmids as appropriate. Correct size of linear amplicons was confirmed by gel electrophoresis, and amplicons were gel purified with a Zymoclean Gel DNA Recovery kit (ZYMO Research, Irvine, CA, USA). Circularized plasmids were constructed from linear fragments via Gibson or GoldenGate assembly, and double-stranded recombineering oligonucleotides were assembled via overlap extension PCR to join upstream and downstream homology regions. E. coli DH10B WT, unless otherwise noted, was transformed with assembled plasmids and grown overnight on LB plates supplemented with the appropriate antibiotic at 30°C or 37°C. Plasmid assembly was confirmed by Sanger sequencing (Genewiz, Chelmsford, MA, USA) of transformant colony PCR product amplified by GoTaq polymerase (Promega, Madison, WI, USA) purified with ExoSAP-IT (Thermo Fisher Scientific, Waltham, MA, USA). Colonies containing sequence-verified plasmids were inoculated into 5.5 mL liquid medium in a 14 mL culture tube, grown overnight, and stocked in 15% (v/v) glycerol, and plasmids were purified from the remaining culture using a PureLink Quick Plasmid Miniprep kit (Invitrogen, Waltham, MA, USA).
10
Cloning and Sequencing of Human OR Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protein-coding region of human OR10A6 and OR2W1 (for accession numbers see Table 2 ) was amplified from human genomic DNA by polymerase chain reaction (PCR), using gene-specific primers (Table S2 ), ligated with T4-DNA ligase (#M1804, Promega, Madison, USA) either MfeI/NotI (#R3589S/ # R0189S, New England Biolabs, Ipswich, UK) or EcoRI/NotI (#R6017/ #R6435, Promega, Madison, USA) into the expression plasmid (#pFN210A SS-HaloTag® CMV-neo Flexi®-Vector, Promega, Madison, USA), and verified by Sanger sequencing (Eurofins Genomics, Ebersberg, Germany) using vector internal primers (Table S3 ).
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!