Restriction and DNA modification enzymes were purchased from New England Biolabs or Fermentas. Plasmid DNA was extracted from E. coli or Sulfolobus cells using an AxyPrep Plasmid Miniprep Kit (Wujiang, China). Polymerase chain reaction (PCR) products were purified by using an Axygen PCR Clean-up Kit, and DNA bands fractionated from the agarose gel were extracted by using an Axygen DNA extraction kit. Total DNA was prepared from Sulfolobus by using an Axygen Genomic DNA Miniprep Kit. The oligonucleotides were synthesized by Invitrogen (Shanghai, China) and were also used for DNA sequencing.
Pcr cleanup kit
Manufactured by Corning
Sourced in United States, China
The PCR cleanup kit is a laboratory product designed to purify and concentrate DNA samples after polymerase chain reaction (PCR) amplification. The kit utilizes a simple and efficient process to remove unwanted components, such as primers, nucleotides, and enzymes, from the PCR reaction mixture, leaving behind the desired DNA fragments.
Automatically generated - may contain errors
Lab products found in correlation
Dna gel extraction kit, by Corning (3 mentions)
T4 dna ligase, by New England Biolabs (2 mentions)
Kod plus high fidelity dna polymerase, by Toyobo (2 mentions)
Phusion dna polymerase, by New England Biolabs (2 mentions)
Miseq platform, by Illumina (2 mentions)
Dl2000 dna marker, by Takara Bio (2 mentions)
Genomic dna miniprep kit, by Corning (1 mentions)
Dna extraction kit, by Corning (1 mentions)
Plasmid miniprep kit, by Corning (1 mentions)
High fidelity dna polymerase, by Toyobo (1 mentions)
Restriction enzyme, by New England Biolabs (1 mentions)
Lightcycler 96 system, by Roche (1 mentions)
Peasy t1 vector, by Transgene (1 mentions)
Tianamp genomic dna kit, by Tiangen Biotech (1 mentions)
Ez dna methylation gold kit, by Zymo Research (1 mentions)
Chip grade protein g agarose beads, by Cell Signaling Technology (1 mentions)
Quantstudio real time pcr, by Thermo Fisher Scientific (1 mentions)
T7en1, by Takara Bio (1 mentions)
Primerstar hs dna polymerase, by Takara Bio (1 mentions)
T vector, by Takara Bio (1 mentions)
30 protocols using pcr cleanup kit
1
Molecular Techniques for DNA Manipulation
2
Plasmid and Chromosomal DNA Extraction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Plasmid DNA was extracted from E. coli by a standard alkaline lysis procedure with a plasmid miniprep kit (Axygen Scientific, Union City, CA, USA). Chromosomal DNA was extracted from Bt cells as previously described (Lereclus et al., 1989 (link)). Restriction enzymes and T4 DNA ligase were used according to the manufacturer's instructions (New England Biolabs, Ipswich, MA, USA). Oligonucleotide primers were synthesized by Sangon (Shanghai, China); all primer sequences are listed in Additional file 5. PCR was performed with high-fidelity DNA polymerase (Toyobo, Osaka, Japan). Amplified fragments were purified with a PCR cleanup kit (Axygen). Digested DNA fragments were separated on 1% agarose gels and extracted using a DNA gel extraction kit (Axygen). All constructs were confirmed by sequencing (Invitrogen, Carlsbad, CA, USA).
3
Standard DNA Manipulation Protocols for E. coli and S. cerevisiae
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
General DNA manipulations in E. coli or S. cerevisiae were performed according to standard methods [64 , 65 ]. Polymerase chain reaction (PCR) was conducted using high-fidelity DNA polymerase KOD plus according to the manufacturer’s instruction (TOYOBO, Japan). The oligonucleotide primers used in this study are listed in Additional file 1 : Table S3. Purification of DNA fragments was performed using PCR Clean-up kit or DNA Gel Extraction kit (Axygen scientific Inc., USA). Total RNA was isolated from yeast cells by using the hot phenol method [65 ]. Gene transcription was analyzed by quantitative real-time PCR (qRT-PCR) using the Quant one-step qRT-PCR kit (SYBR Green) and LightCycler 96 System (Roche, Switzerland). Data were processed by the second-derivative maximum method of LightCycler 96 software SW1.1 with housekeeping gene ACT1 as a control to calculate the relative transcription level of each target gene.
4
TJP1 Promoter DNA Methylation Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genomic DNA was isolated using a TIANamp Genomic DNA kit (Tiangen Biotech, Beijing, China) and was converted using the EZ DNA Methylation-Gold kit (ZYMO Research, Beijing, China). The methylated CpGs in the TJP1 (number NG_003257) promoter were estimated by MethPrimer (https://www.urogene.org/methprimer/ ). The primer pair modified by Primer Premier 5 was 5′-GATTTTATTATTAGTTTTAGTTTTGGTAGT-3′ (forward) and 5′-TAAAAAACTTATCCACTTACTCCTC-3′ (reverse). The amplified fragments were purified using PCR Cleanup kit (Axygen, Corning, NY, USA) and then cloned into pEASY-T1 vector (TransGen Biotech, Beijing, China). The clones were sequenced (BGI Tech, Shenzhen, China) and analyzed data for 20 methylation cites using QUMA (http://quma.cdb.riken.jp/ ).
5
Chromatin Immunoprecipitation of YFP-Tagged Proteins in Arabidopsis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The 2-week-old transgenic Arabidopsis plant overexpressing 35S:YFP or 35S:YFP-AtWRKY20 and the wild-type Col-0 seedlings were used for ChIP assay. Expressing 35S:YFP line was used as a negative control. Three micrograms of seedlings was fixed in 1% formaldehyde for 10 min in a vacuum. Glycine was added to a final concentration of 0.125 M, and the reaction was terminated by incubation for 5 min in a vacuum. Seedlings were rinsed three times with distilled water and frozen in liquid nitrogen for ChIP experiments. ChIP experiments were performed, as described previously, using anti–green fluorescent protein agarose beads (ChromoTek) for immunoprecipitation (38 (link)). The resulting DNA samples were purified with a PCR Cleanup kit (Axygen). DNA fragments were analyzed by qPCR, with the Arabidopsis ACTIN2 promoter as a reference. Enrichments were referred to the 35S:YFP or 35S:YFP-AtWRKY20 against wild-type seedlings. Primers of ChIP assays are listed in table S1.The experiments were repeated with four independent biological replicates and were repeated twice with similar results.
6
Standard DNA Manipulation Protocols in E. coli and S. cerevisiae
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
General DNA manipulations in E. coli or S. cerevisiae were performed according to standard methods38 39 . Polymerase chain reaction (PCR) was conducted using high fidelity DNA polymerase KOD plus according to the manufacturer′s instruction (TOYOBO, Japan). Purification of DNA fragments was performed using PCR Clean-up kit or DNA Gel Extraction kit (Axygen scientific Inc, USA). DNA sequencing was completed by Shanghai Invitrogen Biological Technology CO., LTD. Total RNA was isolated by using the hot phenol method38 . Gene expression was analyzed by qRT-PCR using the Quant one-step qRT-PCR kit (SYBR Green) with ACT1 as a control.
7
ChIP-qPCR Analysis of FOXM1 Binding
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chromatin immunoprecipitation (ChIP) was performed as described previously (Shao et al., 2016 (link)). RAW264.7 cells were treated with PBS or LPS (25 ng/ml) for 6 h, cross-linked with 1% formaldehyde at room temperature for 10 min and quenched by addition of glycine to 125 mM. After centrifugation, cell pellets were washed with cold PBS and lysed in lysis buffer (50 mM HEPES-KOH at pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na deoxycholate, 0.1% SDS, 1 mM PMSF) containing protease inhibitors.
Chromatin was sheared using a Diagenode Bioruptor. Immunoprecipitation was performed using anti-IgG antibody (bsm-33179M-HRP), anti-FOXM1 antibody (ab207298) and ChIP-Grade Protein G Agarose Beads (Cell Signaling Technology). Associated DNA was then purified using a PCR clean-up kit (Axygen) and analyzed by QuantStudio Real-Time PCR (Thermo Fisher). PCR signals from the immunoprecipitated samples were normalized to the input and are presented as the fold change compared with the IgG control. Primer sequences are listed inSupplementary Table S1 .
Chromatin was sheared using a Diagenode Bioruptor. Immunoprecipitation was performed using anti-IgG antibody (bsm-33179M-HRP), anti-FOXM1 antibody (ab207298) and ChIP-Grade Protein G Agarose Beads (Cell Signaling Technology). Associated DNA was then purified using a PCR clean-up kit (Axygen) and analyzed by QuantStudio Real-Time PCR (Thermo Fisher). PCR signals from the immunoprecipitated samples were normalized to the input and are presented as the fold change compared with the IgG control. Primer sequences are listed in
8
T7EN1 Cleavage Assay for Mutation Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Different samples were collected and digested in a lysis buffer (0.4 M NaCl, 2 mM EDTA, 1% SDS, 10 mM Tris-HCl, and 100 mg/ml Proteinase K). The genomic DNA of the sample was extracted from lysate by phenol-chloroform, and recovered by alcohol precipitation. T7EN1 cleavage assay was performed as described by Shen et al.[5 (link)]. Briefly, the targeted fragments were amplified by PrimerSTAR HS DNA polymerase (TaKaRa, DR010A) from the genomic DNA, then purified with a PCR cleanup kit (Axygen, AP-PCR-50). The primers for amplifying targeted fragments were listed in S3 Table . The purified PCR product was denatured and re-annealed in NEBuffer 2 (NEB) using a thermocycler. The PCR products were digested with T7EN1 (NEB, M0302L) for 30 min at 37°C and separated on a 2.5% agarose gel. The PCR products with mutations detected by T7EN1 cleavage assay were sub-cloned into T vector (Takara, D103A). For each sample, the colonies were picked up randomly and sequenced by M13F primer (5’-CGC CAG GGT TTT CCC AGT CAC GAC-3’).
9
Construction of Kozak Consensus Plasmid
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
An expression plasmid containing the Kozak consensus sequence (Kozak, 1987 (link)) was constructed by PCR with specific primers (Supplementary file 1A ). The PCR product was double digested. The digested DNA fragments were purified by PCR Clean-Up Kit (Axygen, Union City, CA) and then subcloned into pcDNA3.0 vector (Invitrogen). The correct insertion was confirmed by DNA sequencing.
10
Analyzing NHEJ-Induced Indel Patterns
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To analyze the indel patterns in NHEJ products, cells were collected after NHEJ induced by Cas9 or nCas9. Briefly, at 72 h after cell transfection with expression plasmids for Cas9- or nCas9-sgRNA, cells were harvested and gDNA was isolated as described above. The targeted regions of less than 300 bp in gDNA were PCR-amplified with primers listed in Supplementary Table 2 . Next-generation sequencing was performed at Novogene Co. Ltd (Beijing). PCR products purified with PCR Clean-up kit (Axygen) were end-repaired, adenylated at 3′ ends, ligated with adapters, purified, and amplified by the second round of PCR to incorporate the P7 and P5 Illumina adapters according to the manufacturer’s protocols (Yeasen, Hieff NGS Ultima DNA Library Prep Kit for Illumina). Sequences were analyzed to determine the editing efficiency and identify the indel pattern at repair junctions using DBS-Aligner as described previously46 (link).
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!