Potential off-target sites were identified using CRISPR RGEN Tools (www.rgenome.net/cas-offinder/ ). For the three CBP targets described above, we computationally selected 33 candidate off-target sites in the rat reference genome (rn5) that were followed by a 5′-NNGRRT PAM with less than five mismatched bases. The genomic region flanking the target sites for each guide sequence was PCR amplified with the following protocol: preheat at 98°C for 30 s, 35 cycles of three-step amplification (98°C for 5 s, 62°C for 10 s, and 72°C for 20 s), and final extension at 72°C for 2 min. The primers for each potential off-target site are listed in table S4. PCR products were purified using DNA Clean-up Kit (CW2301, CWBIO) following the manufacturer’s recommended protocol and sequenced with the MGISEQ-200 (BGI, China). Reads were filtered by an average Phred quality (Q score) > 20 and perfect sequence matches to amplicon forward and reverse primers. Reads from on- and off-target loci were analyzed by first performing alignments against amplicon sequences that included 25 nucleotides upstream and downstream of the target site (a total of 77 bp). Alignments, meanwhile, were analyzed for indels from 5 bp upstream to 5 bp downstream of the target site (37 bp), and indels in this region were discarded with no change among six nucleotides upstream of the PAM sequence (cutting site by SaCas9).
Dna clean up kit
Manufactured by CWBIO
Sourced in China
The DNA Clean-up Kit is a laboratory equipment designed to purify DNA samples from various sources. It efficiently removes contaminants and impurities, allowing for the recovery of high-quality DNA for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Multif seamless assembly mix, by ABclonal (2 mentions)
Dpni, by Takara Bio (2 mentions)
Primestar max dna polymerase, by Takara Bio (2 mentions)
Pureplasmid mini kit, by CWBIO (1 mentions)
Onetaq 2 master mix, by New England Biolabs (1 mentions)
Clonexpress 2 one step cloning kit, by Vazyme (1 mentions)
Q5 high fidelity 2 master mix, by New England Biolabs (1 mentions)
Pgem t easy vector, by Takara Bio (1 mentions)
Ex taq, by Takara Bio (1 mentions)
Tianamp genomic dna kit, by Tiangen Biotech (1 mentions)
Taq dna polymerase, by Transgene (1 mentions)
All in one first strand cdna synthesis supermix for qpcr, by Transgene (1 mentions)
Jm109 de3, by Promega (1 mentions)
Fastpure plasmid mini kit, by Vazyme (1 mentions)
Xbai hf, by New England Biolabs (1 mentions)
Cell rna kit, by Yeasen (1 mentions)
Hindiii hf, by New England Biolabs (1 mentions)
Ecori hf, by New England Biolabs (1 mentions)
T4 ligase enzyme, by New England Biolabs (1 mentions)
Bamhi hf, by New England Biolabs (1 mentions)
8 protocols using dna clean up kit
1
Comprehensive Off-Target Analysis for CRISPR Editing
2
Efficient Bacterial DNA and Plasmid Isolation
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The isolation of genomic DNA and plasmid preparation using E. coli or S. aureus were performed with a bacterial genomic DNA kit and PurePlasmid Mini Kit, respectively (CwBIO, Jiangsu, China). Oligonucleotide primers were synthesized by Sangon Biotech (Shanghai, China). All primers used in this study are listed in Supplementary Table 2 . For analytical purposes, PCRs were performed using OneTaq 2 × Master Mix (NEB, Ipswich, England). PCRs for plasmid construction were performed using Q5 High-Fidelity 2 × Master Mix from NEB according to the manufacturers’ instructions. The PCR products were purified using the DNA Clean-up Kit (CwBIO, Jiangsu, China). For cloning or plasmid construction, the plasmid was linearized by the related restriction enzymes from NEB. Cloning was performed using the ClonExpress II One Step Cloning Kit (Vazyme, Nanjing, China), which is based on homologous recombination.
3
Seamless Cloning of Plasmids in E. coli and B. subtilis
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In this study, all plasmids were constructed by the seamless cloning technique, based on Gibson assembly, reported previously (52 (link)). The high-fidelity PrimeSTAR Max DNA polymerase (TaKaRa Bio Inc., Japan) was used for the amplification of DNA fragments required for plasmid construction. The PCR products were treated with DpnI (TaKaRa Bio Inc.) and then cleaned up with a DNA clean-up kit (CWBIO, China) to remove the templates. The ABclonal MultiF seamless assembly mix (ABclonal Technology Co. Ltd., China) was used for the seamless ligation of purified fragments. The reaction product was directly transformed into E. coli JM109 competent cells. The constructed plasmids were then extracted and transformed into B. subtilis 168 or WB800 cells. The primers used in this study are listed in Table S2 in the supplemental material. For the in vitro assembly of amplified fragments, 15- to 3-bp-long homologous sequences were designed in the primers.
4
Genomic DNA Extraction and Defensin Gene Amplification from Scorpion Martensii
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Genomic DNA was isolated as previously described [31 (link)]. Simply intact individuals of the scorpion M. martensii were washed three times with 75% ethanol and grounded to fine powder in liquid nitrogen. Then genomic DNA extraction was performed using TIANamp Genomic DNA Kit (Tiangen, China) according to the manufacturer’s instructions.
Primers (Supplementary Table S1) from 5′-UTR and 3′-UTR regions of the predicted defensin genes from the scorpion M. martensii genome were picked up to amplify the corresponding genomic DNA by PCR. Amplification was performed with one cycle of 5 min at 95°C, 30 cycles of 40 s at 95°C, 40 s at 58°C, 150 s at 72°C, and a final cycle of 10 min at 72°C using Ex Taq (TaKaRa, China). PCR products were purified using the DNA Clean-up Kit (CWBio, China) and ligated to pGEM-T Easy Vector (TaKaRa, China). Sequencing was performed by Tsingke Biological Technology.
Primers (Supplementary Table S1) from 5′-UTR and 3′-UTR regions of the predicted defensin genes from the scorpion M. martensii genome were picked up to amplify the corresponding genomic DNA by PCR. Amplification was performed with one cycle of 5 min at 95°C, 30 cycles of 40 s at 95°C, 40 s at 58°C, 150 s at 72°C, and a final cycle of 10 min at 72°C using Ex Taq (TaKaRa, China). PCR products were purified using the DNA Clean-up Kit (CWBio, China) and ligated to pGEM-T Easy Vector (TaKaRa, China). Sequencing was performed by Tsingke Biological Technology.
5
Molecular Cloning and Cell Culture Protocol
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High fidelity restriction endonucleases BamHI-HF, HindIII-HF, EcoRI-HF, XbaI-HF, and T4 ligase enzyme were purchased from NEB (New England Biolabs, MA, USA). Bacteria strain DH10B, BL21(DE3), Taq DNA polymerase, and All-in-One First-Strand cDNA Synthesis SuperMix for qPCR were purchased from Transgen, and JM109(DE3) was purchased from Promega (Promega, WI, USA). pT7Oi and pT7Om vectors were already constructed by our group. psilence-2.1-U6-Hygro vector was purchased from Beijing Rambolide Trading Co. DNA fragments were purified with AxyPrep DNA Gel Extraction Kit (Axygen) or DNA Clean-up Kit (Cwbio), and plasmids were extracted by FastPure Plasmid Mini Kit (Vazyme) or Endo-Free Plasmid Mini Kit I (Omega). Cell RNA Kit, BCA protein quantification kit, and Hieff Trans liposomal transfection reagent were purchased from Yeasen. ProtLytic Protein Lysis and Sample Loading was purchased from New Cell & Molecular biotech Co. Human embryonic kidney 293T (HEK293T) was a gift from Professor Yao Shaohua’s laboratory. Michigan Cancer Foundation-7 (MCF-7) and human hepatocellular carcinomas (Hep G2) were purchased from ATCC (American Type Culture Collection, VA, USA).
6
Cloning Sso7d and T7C565-883 Fragments
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First round PCR: 1 ng of KOD-S7 plasmid and Rosetta (DE3) genomic DNA were used as template, and S7dF & S7dR, T7cF & T7cDrdR were used as primer pairs for amplification of the Sso7d DNA fragment and T7C565-883 DNA fragment, respectively. The PCR extension time was 30 s. The PCR products were purified by DNA Clean-up Kit (CWBIO). Second round PCR: 1 ng of both the purified Sso7d DNA fragment and T7C565-883 DNA fragment were mixed with 1 ng of pLysS, and 0.4 μM of primers S7dR and T7cF were used. The PCR extension time was 2 min.
7
Chromatin Immunoprecipitation (ChIP) Assay
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The ChIP assay was prepared using a ChIP assay kit (Beyotime; P2078) following the manufacturer's instructions. For the BRD7 ChIP assay, the chromatin immunoprecipitations were performed with ChIP formulated BRD7 (D9K2T) rabbit monoclonal antibody (Mab) (CST; no.14910). DNA was purified by DNA clean-up kit (CWbiotech). ChIP libraries were prepared using the NEBNext UltraTMII DNA Library preparation kit (no. E7645S) complemented with NEXTflex DNA barcodes from Bioo Scientific. Ten nanograms of DNA was used as starting material for the input and immunoprecipitation group samples. Libraries were amplified on the thermocycler. Postamplification libraries were size selected at 250 to 450 bp in length using AMPure XP Beads (Beckman Coulter; no. A63881) from Beckman Coulter. ChIP Libraries were validated using Qubit kit and then run on the Illumina Novoseq6000 sequencer.
8
Seamless Cloning of Plasmids in E. coli and B. subtilis
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In this study, all plasmids were constructed by the seamless cloning technique, based on Gibson assembly, reported previously (52 (link)). The high-fidelity PrimeSTAR Max DNA polymerase (TaKaRa Bio Inc., Japan) was used for the amplification of DNA fragments required for plasmid construction. The PCR products were treated with DpnI (TaKaRa Bio Inc.) and then cleaned up with a DNA clean-up kit (CWBIO, China) to remove the templates. The ABclonal MultiF seamless assembly mix (ABclonal Technology Co. Ltd., China) was used for the seamless ligation of purified fragments. The reaction product was directly transformed into E. coli JM109 competent cells. The constructed plasmids were then extracted and transformed into B. subtilis 168 or WB800 cells. The primers used in this study are listed in Table S2 in the supplemental material. For the in vitro assembly of amplified fragments, 15- to 3-bp-long homologous sequences were designed in the primers.
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