Blunt end miniTol2-KanR fragments were generated by PCR with platinum Pfx DNA polymerase (Invitrogen) with forward primer Tol2-mini5′ (5′- CAGAGGTGTAAAGTACTTGA-3′), reverse primer Tol2-mini3′ (5′- CAGAGGTGTAAAAAGTACTC-3′) and a template (pTol2miniKan(-)amp). PCR product was treated with DpnI to get rid of the template before purified by QIAquick PCR purification kit (Qiagen). Freshly prepared plasmids and PCR products were used in the assay. In a 20 μL reaction, 0.5 pMol plasmid pGL, corresponding PCR fragments and 27 pMol His-Tol2 (~2 μg) were mixed in MOPS buffer (25 mM MOPS, pH 7.0; 1 mM MnCl2; 50 mM NaCl; 5 % glycerol; 2 mM DTT; 100 ng/ul BSA). The reaction was incubated for 2 h at 30 °C. DNA was phenol-chloroform extracted using Maxtract High Density columns (Qiagen) and resuspended in 5 μL TE buffer. 1 μL or 5 μL DNA was used to transform Top10 competent cells (Invitrogen). The number of colonies grew on Amp-resistant plates was used to calculate the total number recovered plasmids, and the number of colonies that grew on Kan-resistant plates was used to calculate the number of plasmid with KanR integration. MOPS buffer with MgCl2 or no added cations were tested as well, and the integration activity was modestly higher with MnCl2.
Maxtract high density columns
Manufactured by Qiagen
Maxtract High Density columns are a type of laboratory equipment designed for the extraction and purification of nucleic acids, such as DNA and RNA, from various biological samples. The core function of these columns is to efficiently separate and concentrate the desired nucleic acid molecules from complex sample matrices, allowing for their subsequent analysis or downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Simplechip enzymatic chromatin ip kit, by Cell Signaling Technology (2 mentions)
Top10 competent cell, by Thermo Fisher Scientific (1 mentions)
Platinum pfx dna polymerase, by Thermo Fisher Scientific (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Nebnext dna library kit, by New England Biolabs (1 mentions)
Scriptcap m7g capping system, by CellScript (1 mentions)
Ribomax large scale rna production system, by Promega (1 mentions)
Easysep mouse cd4 cd25 regulatory t cell isolation kit 2, by STEMCELL (1 mentions)
Nebnext ultra 2 dna library prep kit, by New England Biolabs (1 mentions)
Cfx384 detection system, by Bio-Rad (1 mentions)
Qbaseplus, by CellCarta (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Merck Group (1 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
5 protocols using maxtract high density columns
1
Tol2 Integration Assay Protocol
2
Bcl11b ChIP-Seq in CD4+ T-cells
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ChIP-Seq against Bcl11b was performed on CD4+ T-cells using the SimpleChIP Enzymatic Chromatin IP Kit from Cell Signaling Technologies (#9003), following their recommended protocol up to the library preparation with the following alterations: (1) 107 cells were used in place of 4 × 107; (2) formaldehyde fixation was performed in 1 mL; (3) Buffer A and Buffer B steps were all performed in 1 mL; (4) 2 μl of 1:10 diluted Micrococcal nuclease was determined to give the optimum digestion results by titration; (5) cocktail of two anti-Bcl11b antibodies were used (8 μg ab18465 and 3 μg of CST #12120 per ChIP); (6) final DNA purification was performed by phenol:chloroform:isoamyl alcohol extraction with MaXtract high density columns (Qiagen #129046). Libraries were prepared using NEBNext DNA Library Kit (NEB, #E7645), with quality being determined as above. Libraries were run as SE75 on a NextSeq 500. Fastq analysis performed as with ATAC-Seq, but with single-end rather than paired-end settings.
3
Optimized Tc24 mRNA Production for Protein Expression
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Tc24 sequences were cloned into pTNT (Promega) to facilitate mRNA transcription. Codon usage was optimized to improve ribosome binding and expression efficacy. A KOZAK sequence (GCCRCCAUGG) was added ahead of the Tc24 ORF to assist in the initiation of the translation process. The plasmid was cloned in Escherichia coli. To produce RNA, 200 μg of plasmid encoding Tc24 were first linearized using BamHI. The plasmid was then purified on MaXtract High-Density columns (Qiagen) and by precipitation in ethanol and quantified by absorbance at 260 nm on a Nanodrop spectrophotometer. The linearized plasmid was stored at -20 °C until use. Subsequently, RNA was transcribed using RiboMAX Large Scale RNA Production Systems (Promega), replacing uridine for modified 5-Methoxy-UTP (Trilink). These RNAs were purified by precipitation in ammonium acetate and capped using ScriptCap™ m7G Capping System (CellScript) to enhance cell RNA stability and translation. The mRNA was quantified by absorbance at 260 nm on a Nanodrop spectrophotometer. All RNAs were stored at -80 °C until use. The integrity, quantity, and quality of the transcribed RNA were assessed through denaturing agarose gel electrophoresis. Protein translation was verified by in vitro translation in a mouse dendritic cell line (DC 2.4, Millipore Sigma) followed by SDS-gel electrophoresis and western blotting (Supplement data 1) .
4
Bcl11b ChIP-seq in Treg cells
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Treg cells were isolated from the spleens and peLNs of naïve mice and enriched using the EasySep Mouse CD4+ CD25+ Regulatory T Cell Isolation Kit II (#18783, STEMCELL). Human Treg cells were isolated from PBMCs by sorting CD4+ CD25+ CD127− cells. Bcl11b ChIP-seq was performed on Treg cells using the SimpleChIP Enzymatic Chromatin IP Kit from Cell Signaling Technology (CST #9003), following their recommended protocol up to the library preparation with the following alterations: (i) 107 cells were used in place of 4 × 107, (ii) formaldehyde fixation was performed in 1 ml, (iii) buffer A and B steps were all performed in 1 ml, (iv) cells were sheared in ChIP buffer to an average of 200 to 1000 base pairs using a Diagenode Bioruptor Pico (B01060002), (v) a cocktail of three anti-Bcl11b antibodies were used (4 μg of ab18465, 4 μg of CST #12120, and 4 μg of Bethyl A300-385 per ChIP), and (vi) final DNA purification was performed by phenol:chloroform:isoamyl alcohol extraction and MaXtract high-density columns (#129046, Qiagen). Libraries were prepared using NEBNext Ultra II DNA Library Prep Kit (#E7645S, New England Biolabs) and sequenced as SE75 on a NextSeq500.
5
Quantitative PCR Analysis of Gene Expression
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Total RNA was isolated with TRIzol reagent (Invitrogen) using Maxtract high-density columns (Qiagen, ref. 1038988). Isolated RNA was treated with DNAse I (Sigma-Aldrich, D5025) and reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). For mouse samples, quantitative PCR (qPCR) was performed with SYBR Green PCR master mix (Applied Biosystem) in a CFX384 detection system (Bio-Rad). Primers used in this study are listed in Supplementary Table 1 . Data were normalized to the expression levels of 36b4 and cyclophilin mRNA within individual samples. For human samples, qPCR was performed using TaqMan probes for human MMP17 (Hs00211754_m1) and normalized to the expression levels of TBP (Hs99999910_m1). All samples were analyzed in triplicate and RNA levels (CNRQ; calibrated normalized relative quantity) were calculated with Biogazelle qBase PLUS.
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