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Dna ladder

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A DNA ladder is a molecular weight standard used to determine the size of DNA fragments in gel electrophoresis. It consists of a mixture of DNA fragments of known sizes that can be used as a reference to estimate the size of unknown DNA samples.

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Lab products found in correlation

Deoxyribonucleotides, by New England Biolabs (4 mentions) Restriction enzyme, by New England Biolabs (3 mentions) Dna modifying enzymes, by New England Biolabs (3 mentions) Taq dna polymerase, by New England Biolabs (2 mentions) Green gotaq reaction buffer, by Promega (2 mentions) Exo sap treated, by Thermo Fisher Scientific (2 mentions) Nucleic acid purification kits, by Macherey-Nagel (2 mentions) Oligonucleotides, by Eurofins (2 mentions) Restriction endonuclease, by New England Biolabs (2 mentions) Dna polymerase, by New England Biolabs (2 mentions) T4 dna ligase, by New England Biolabs (2 mentions) Ammonium sulfate, by Merck Group (2 mentions) Oligonucleotide primers, by Thermo Fisher Scientific (2 mentions) Gibson assembly master mix, by New England Biolabs (2 mentions) Q5 high fidelity 2x master mix, by New England Biolabs (2 mentions) Gateway lr clonase 2 enzyme mix, by Thermo Fisher Scientific (2 mentions) Qiaquick gel extraction kit, by Qiagen (1 mentions) Dna extraction kit, by Qiagen (1 mentions) Gblock fragment, by Integrated DNA Technologies (1 mentions) Kanamycin sulfate, by Merck Group (1 mentions)

Close spelling variants of this product

100 bp DNA ladder (70 citations) 1 kb DNA ladder (32 citations) 1 kb Plus DNA Ladder (21 citations) Low molecular weight DNA ladder (20 citations) 2-Log DNA Ladder (16 citations) Quick-Load 100 bp DNA ladder (13 citations) 50 bp DNA ladder (11 citations) Quick-Load Purple 100 bp DNA Ladder (8 citations) Quick Extend DNA ladder (7 citations) PBR322 DNA-MspI Digest ladder (4 citations)

35 protocols using dna ladder

1

Generating Transgenic C. elegans with Altered sid-1 Expression

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Worms carrying the extrachromosomal and integrated Psid-1::gfp arrays were generated previously (Winston et al., 2002 (link)). To generate Psid-1 worms, the sid-1 promoter was amplified from N2 genomic DNA (primers: 5′-GGTCATGAGAGGGTCGAGAG-3′, 5′-GGAAAAATGAGGAGTTTTAATTTC-3′) and gel purified (QIAquick Gel Extraction Kit, Qiagen, 28704). To make complex extrachromosomal array lines, the germline of N2 (wild-type) worms was injected with 0.1–75ng/μl Psid-1, 15ng/μl pHC183 (myo-3::dsRed2) (Winston et al., 2002 (link)) and 25ng/μl DNA ladder (New England Biolabs, N3232S).
The Psid-1(piRNA-4A) fragment was amplified from genomic DNA in two pieces using a site directed mutagenesis strategy to introduce the appropriate mutations (See Table S3 for primers) and cloned (pHC516). Psid-1(piRNA-4A) fragments were amplified, gel purified and injected at a concentration of 50ng/μl or 1ng/μl into N2 or prg-1(n4357) worms with 15ng/μl pHC183 (myo-3::dsRed2) (Winston et al., 2002 (link)) and 25ng/μl DNA ladder (New England Biolabs, N3232S).
Minkina O, & Hunter C.P. (2017). Stable heritable germline silencing directs somatic silencing at an endogenous locus. Molecular cell, 65(4), 659-670.e5.
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2

Cloning and Expression of Recombinant Proteins

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pET24+ vectors were purchased from Novagen (Madison, WI). gBlock fragments were purchased from Integrated DNA Technologies (Coralville, IA). Ligation enzymes, restriction enzymes, and DNA ladders were purchased from New England BioLabs (Ipswich, MA). BL21 (DE3) chemically competent Escherichia coli cells were purchased from Bioline (Taunton, MA). All E. coli cultures were grown in Terrific Broth (TB) media purchased from VWR International (Radnor, PA). Kanamycin sulfate was purchased from EMD Millipore (Billerica, MA). Protein expression was induced with isopropyl β-d-1-thiogalactopyranoside (IPTG) from Gold Biotechnology (St. Louis, MO). All salts were purchased from Sigma-Aldrich (St. Louis, MO). 1× Phosphate-buffered saline (PBS) tablets (10 mM phosphate buffer, 140 mM NaCl, 3 mM KCl, pH 7.4 at 25 °C) were purchased from EMD Millipore (Billerica, MA). DNA extraction kits and DNA gel purification kits were purchased from Qiagen, Inc. (Germantown, MD). Whatman Anotop sterile syringe filters (0.02 μm) were purchased from GE Healthcare Life Sciences (Pittsburgh, PA).
Navarro L.A., Ryan J.J., Dzuricky M., Gradzielski M., Chilkoti A, & Zauscher S. (2021). Microphase Separation of Resilin-like and Elastin-like Diblock Copolypeptides in Concentrated Solutions. Biomacromolecules, 22(9), 3827-3838.
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3

Bacterial Growth Conditions and Reagents

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Bacterial strains used in this study are listed in Table 1. Glycerol, Tween 80, kanamycin and IPTG were purchased from SIGMA, USA. Restriction enzymes, Taq DNA polymerase, DNA ladders were purchased from New England Biolabs, USA. Hygromycin B was obtained from Roche, Fusion polymerase from Finnzymes. Pristinamycin was obtained from Sanofi Aventis and the P1 component of Pristinamycin was purified in-house as described earlier [26 (link)]. Luria Bertani (LB) broth and LB agar were used to grow E. coli. Middlebrook 7H9 (DIFCO) supplemented with 0.2% Glycerol (v/v), 0.05% Tween 80 (w/v) and albumin-dextrose was used for growing broth cultures of mycobacteria and Middlebrook 7H11 (DIFCO) for measurement of colony forming units (CFU). Bacterial cultures were supplemented with antibiotics, IPTG and P1 as required.
Ravishankar S., Ambady A., Swetha R.G., Anbarasu A., Ramaiah S, & Sambandamurthy V.K. (2016). Essentiality Assessment of Cysteinyl and Lysyl-tRNA Synthetases of Mycobacterium smegmatis. PLoS ONE, 11(1), e0147188.
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4

Genomic DNA Extraction and PCR Analysis of CAG Repeats

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Genomic DNA was isolated from CanR clones using a previously described method64 . Cells were resuspended in 1.5 µL of 0.5 mg/mL lyticase solution [0.9 M Sorbitol, 0.1 M EDTA (pH 7.4)] in microplates, incubated at 37°C for 15 min, then resuspended in 50 µL of water. The samples were incubated at 100°C for 5 min and centrifuged at 2,500 × g for at least 2 min. For PCR analysis of CAG repeat length, reactions included 1× Green GoTaq reaction buffer (M7911, Promega), 0.16 mM dNTP mix, 0.8 µM of each primer, 0.5 units of Taq DNA polymerase (SibEnzyme or Thermo Scientific), and 1 µL of the DNA supernatant in a 12.5 µL total reaction volume. Primers JK316 and JK317 result in a 544 bp product for (CAG)140. Primers JK318 and JK319 result in a 625 bp product for (CAG)140 (Figure 1B). Primers JK153 and JK171 result in a 462 bp product for (CAG)140 and 321 bp (CAG)93. 10 µL PCR products were run on 1.5% agarose in 0.5× TBE alongside 50 bp and 100 bp DNA ladders (NEB). PCR products were sized using TotalLab Quant software for 1D DNA gels.
The same PCR method was used to generate products for sequencing the CAG repeats (FlankL and CanF) or the CAN1 gene (JK167 and JK168, JK169 and JK170). PCR products were Exo-SAP treated (Affymetrix) and sequenced by Eton Bioscience or University of Chicago Sequencing Core).
Kim J.C., Harris S.T., Dinter T., Shah K.A, & Mirkin S.M. (2016). The role of break-induced replication in large-scale expansions of (CAG)n•(CTG)n repeats. Nature structural & molecular biology, 24(1), 55-60.
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5

Purification and Biochemical Characterization of Sirtuins

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All restriction enzymes, DNA-modifying enzymes, and DNA ladders were obtained from New England Biolabs. Plasmid pETM-41 and TEV protease were kindly provided by Dr Amit Sharma (ICGEB, New Delhi). Protein markers were obtained from Thermo Fisher Scientific (USA). nicotinamide Adenine Dinucleotide [Adenylate-32P] (800Ci/mmol) was purchased from American Radiolabeled Chemicals (USA). Ni2+-NTA agarose and amylose resin were purchased from Qiagen and New England Biolabs, respectively. Sirtinol, nicotinamide, cambinol, and Ex-527 were obtained from Sigma-Aldrich (USA). SIRT1 Fluorometric Drug Discovery Kit and SIRT5 Fluorometric Drug Discovery Kit were procured from Enzo Life Sciences (USA). Other materials used in this study were of analytical grade and were commercially available.
Mittal N., Muthuswami R, & Madhubala R. (2017). The mitochondrial SIR2 related protein 2 (SIR2RP2) impacts Leishmania donovani growth and infectivity. PLoS Neglected Tropical Diseases, 11(5), e0005590.
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6

Reagents and Suppliers for Molecular Biology

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Chemicals were purchased from Sigma-Aldrich (Dorset, UK) and ForMedium (Norfolk, UK). DNA modifying enzymes, deoxyribonucleotides, and DNA ladders were purchased from New England Biolabs (Hitchin, UK), Thermo Fisher Scientific (Loughborough, UK), and Agilent Technologies (Cheadle, UK). Nucleic acid purification kits were purchased from Machery-Nagel (Düren, Germany) and Omega Bio-tek (Norcross, USA). All oligonucleotides were synthesized by Eurofins Genomics (Ebersberg, Germany), and summarized in Supplementary Table S1.
Gonzalez-Perez D., Ratcliffe J., Tan S.K., Wong M.C., Yee Y.P., Nyabadza N., Xu J.H., Wong T.S, & Tee K.L. (2021). Random and combinatorial mutagenesis for improved total production of secretory target protein in Escherichia coli. Scientific Reports, 11, 5290.
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7

Purification of LdThrRS Enzyme

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All restriction enzymes and DNA ladders were acquired from New England Biolabs (NEB) (MA, USA). The expression vector pET30a was obtained from Novagen (Germany). Ni2+-NTA (nitrilotriacetic acid) agarose beads were obtained from Qiagen (USA). Hygromycin, paromomycin and zeocin were acquired from Sigma Aldrich (Germany). Protein ladders were obtained from Fermentas. Borrelidin was purchased from Abcam (Cambridge, UK). The in vitro tRNA transcription kit was obtained from Invitrogen (CA, USA). The anti-LdThrRS antibody was commercially synthesized in rabbits by Merck (Germany).
Chadha S., Vijayan R., Gupta S., Munde M., Gourinath S, & Madhubala R. (2018). Genetic manipulation of Leishmania donovani threonyl tRNA synthetase facilitates its exploration as a potential therapeutic target. PLoS Neglected Tropical Diseases, 12(6), e0006575.
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8

DNA Enzyme Purchases from NEB

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All enzymes, deoxyribonucleotides, and DNA ladders were purchased from New England Biolabs Ltd. (Hitchin, UK).
Jajesniak P., Tee K.L, & Wong T.S. (2019). PTO-QuickStep: A Fast and Efficient Method for Cloning Random Mutagenesis Libraries. International Journal of Molecular Sciences, 20(16), 3908.
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9

Molecular Cloning Techniques Utilizing Phusion and TOPO TA

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The Template Generation System II Kit from Finnzymes was used to perform the transposition reactions. TOPO TA cloning Kit and pUC19 vector (2.68 kb) were purchased from Invitrogen. Phusion High Fidelity DNA Polymerase from New England Biolabs (NEB) was used in all PCR reactions in Phusion buffer (20 mM Tris-HCl pH 8.8, 20 mM KCl, 20 mM, (NH4)2SO4, 2 mM MgCl2, 100 μg/ml bovine serum albumin, 1.25 M betaine, 0.1% Triton X-100). Deoxynucleoside triphosphate (dNTP) solutions and restriction enzymes were purchased from NEB; primers were purchased from Integrated DNA Technologies, and ethylenediaminetetraacetic acid (EDTA) (0.5 M, pH 8.0) was purchased from AccuGENE. Gel Extraction Kit and PCR Purification Kit (QIAGEN) were used according to manufacturer instructions. DNA concentrations were determined by measuring absorbance at 260 nm on a Nano Drop spectrophotometer 2000c (Thermo Scientific). A total of 10X Tris-Borate EDTA (TBE) buffer from National Diagnostics contained 0.89 M Tris Borate pH 8.3 and 20 mM EDTA disodium salt. Gel electrophoresis was conducted in 0.5X Tris-Borate EDTA buffer (TBE). DNA ladders were purchased from NEB.
Morelli A., Cabezas Y., Mills L.J, & Seelig B. (2017). Extensive libraries of gene truncation variants generated by in vitro transposition. Nucleic Acids Research, 45(10), e78.
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10

Genomic DNA Extraction and PCR Analysis of CAG Repeats

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Genomic DNA was isolated from CanR clones using a previously described method64 . Cells were resuspended in 1.5 µL of 0.5 mg/mL lyticase solution [0.9 M Sorbitol, 0.1 M EDTA (pH 7.4)] in microplates, incubated at 37°C for 15 min, then resuspended in 50 µL of water. The samples were incubated at 100°C for 5 min and centrifuged at 2,500 × g for at least 2 min. For PCR analysis of CAG repeat length, reactions included 1× Green GoTaq reaction buffer (M7911, Promega), 0.16 mM dNTP mix, 0.8 µM of each primer, 0.5 units of Taq DNA polymerase (SibEnzyme or Thermo Scientific), and 1 µL of the DNA supernatant in a 12.5 µL total reaction volume. Primers JK316 and JK317 result in a 544 bp product for (CAG)140. Primers JK318 and JK319 result in a 625 bp product for (CAG)140 (Figure 1B). Primers JK153 and JK171 result in a 462 bp product for (CAG)140 and 321 bp (CAG)93. 10 µL PCR products were run on 1.5% agarose in 0.5× TBE alongside 50 bp and 100 bp DNA ladders (NEB). PCR products were sized using TotalLab Quant software for 1D DNA gels.
The same PCR method was used to generate products for sequencing the CAG repeats (FlankL and CanF) or the CAN1 gene (JK167 and JK168, JK169 and JK170). PCR products were Exo-SAP treated (Affymetrix) and sequenced by Eton Bioscience or University of Chicago Sequencing Core).
Kim J.C., Harris S.T., Dinter T., Shah K.A, & Mirkin S.M. (2016). The role of break-induced replication in large-scale expansions of (CAG)n•(CTG)n repeats. Nature structural & molecular biology, 24(1), 55-60.
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