Cas9 mRNA was transcribed from the XbaI-linearized pJET1.2-SpCas9 vector61 (link) using the HiScribe T7 ARCA mRNA Kit (with tailing) (NEB, Ipswich, MA, USA); mRNA was purified using LiCl precipitation. Oligonucleotides corresponding to the human TP53- or GFP-targeting portion of sgRNAs were cloned into pX330-U6-Chimeric_BB-CBh-hSpCas9.62 (link) sgRNA-coding sequences were amplified with primers containing a T7 promoter sequence at the 5′ end of the forward primer, purified using the Clean-Up Concentrator kit (A&A Biotechnology), in vitro transcribed using the TranscriptAid T7 High Yield Transcription Kit (Thermo Scientific), and then purified by LiCl precipitation. A549 cells were electroporated with 5 μg of Cas9 mRNA in electroporation buffer using Gene Pulser II (Bio-Rad Laboratories) under the following conditions: one million cells in 400 μL electroporation buffer in a 0.4-cm gap cuvette and pulse voltage and capacitance: 400 V and 700 μF. After 4 h, electroporation was repeated using the same procedure, but with 1 μg of TP53- or GFP-targeting sgRNAs instead of Cas9 mRNA. The cells were allowed to regenerate in culture (for about 3 days), and the electroporation was repeated with another TP53-targeting sgRNA (three in total). Sequences of oligonucleotides used for sgRNA cloning and for amplification of sgRNA for in vitro transcription are available in Table S5 .
Clean up concentrator kit
Manufactured by A&A Biotechnology
Sourced in Poland
The Clean-up Concentrator kit is a laboratory equipment designed for the concentration and purification of biological samples. It utilizes a centrifugal mechanism to efficiently remove unwanted materials and concentrate the desired analytes.
Automatically generated - may contain errors
Lab products found in correlation
Miseq apparatus, by Illumina (3 mentions)
Plasmid midi ax, by A&A Biotechnology (3 mentions)
Plasmid mini kit, by A&A Biotechnology (3 mentions)
Kapa library preparation kit reagents, by Roche (3 mentions)
Miseq reagent kit v3 600 cycle, by Illumina (2 mentions)
Transcriptaid t7 high yield transcription kit, by Thermo Fisher Scientific (1 mentions)
Hiscribe t7 arca mrna kit, by New England Biolabs (1 mentions)
Gene pulser 2, by Bio-Rad (1 mentions)
Miseq reagent kit v3, by Illumina (1 mentions)
Genejet plasmid miniprep kit, by Thermo Fisher Scientific (1 mentions)
7 protocols using clean up concentrator kit
1
CRISPR-Cas9 Mediated Gene Editing in A549 Cells
2
Splicing Minigene Construct Generation
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EGFP-MBNL1-41, EGFP-MBNL2-38, Atp2a1-WT and Atp2a1-D minigenes were previously described17 (link), 57 (link). Mouse Ank2 and mutAnk2 as well as human ANK2 and mutANK2 splicing minigenes were generated by cloning DNA oligonucleotides between NotI and SalI restriction sites in Atp2a1-D minigene. 120 bp DNA oligonucleotides contained selected TGCT(N)3TGCT(N)13–18TGCT/C or mutated G GCT(N)3TGA T(N)13–18TGT C sequences (substitutions are underlined) as well as NotI and SalI restriction sites at 5’ and 3’ ends respectively. DNA oligonucleotide sequences are listed in the key resources table. The complementary single-stranded DNA oligonucleotides (100 μM) were annealed in Annealing Buffer (10 mM Tris, pH 7.5, 50 mM NaCl, 1 mM EDTA) at 95°C for 5 minutes followed by cooling to 25°C for 45 minutes. Annealed oligonucleotides were digested with NotI and SalI restriction enzymes (New England Biolabs), purified using Clean-Up Concentrator Kit (A&A Biotechnology), and ligated. The design of the hybrid Atp2a1 minigenes preserves RNA structures within a thermodynamically stable region at the Atp2a1 insertion site57 (link). Final splicing minigenes were tested by Sanger sequencing.
3
Acidovorax sp. A1169 Genome Sequencing
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Genomic DNA was isolated by the CTAB method (Wilson 2001 ). Plasmid isolation was performed with the Plasmid Midi AX or the Plasmid Mini kits (A&A Biotechnology) while DNA purification was conducted with the Clean-up Concentrator kit (A&A Biotechnology) according to manufacturer instructions. The genome of Acidovorax sp. A1169 was sequenced using an Illumina MiSeq apparatus (Illumina Inc., USA). The Illumina paired-end sequencing library construction was performed with 1 μg of post-nebulized DNA extract and the KAPA Library Preparation Kit reagents (KAPA Biosystems, USA), according to the manufacturer’s instructions. The library was pooled and sequenced on a MiSeq platform using the 600-cycle MiSeq reagent Kit v.3 (Illumina, USA). Sequence quality metrics were assessed using FASTQC (Andrews 2010 ).
4
Genomic DNA Extraction and Illumina Sequencing
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Genomic DNA was isolated by the CTAB method (Wilson 2001 ). Plasmid isolation was performed with the Plasmid Midi AX or the Plasmid Mini kits (A&A Biotechnology) while DNA purification was conducted with the Clean-up Concentrator kit (A&A Biotechnology) according to manufacturer’s instructions. The genomes of Acidovorax sp. A1169 and Collimonas sp. A2191 were sequenced using an Illumina MiSeq apparatus (Illumina Inc., USA). The Illumina paired-end sequencing library construction was performed with 1 μg of post-nebulized DNA extract and the KAPA Library Preparation Kit reagents (KAPA Biosystems, USA), according to the manufacturer’s instructions. The library was pooled and sequenced on a MiSeq platform using the 600-cycle MiSeq reagent Kit v.3 (Illumina, USA). Sequence quality metrics were assessed using FASTQC (Andrews 2010 ).
6
Genomic DNA Isolation and Illumina Sequencing
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Genomic DNA was isolated by the CTAB method (Wilson 2001 (link)). Plasmid isolation was performed with the Plasmid Midi AX or the Plasmid Mini kits (A&A Biotechnology) while DNA purification was conducted with the Clean-up Concentrator kit (A&A Biotechnology) according to manufacturer instructions. The genome of Acidovorax sp. A1169 was sequenced using an Illumina MiSeq apparatus (Illumina Inc., USA). The Illumina paired-end sequencing library construction was performed with 1 μg of post-nebulized DNA extract and the KAPA Library Preparation Kit reagents (KAPA Biosystems, USA), according to the manufacturer’s instructions. The library was pooled and sequenced on a MiSeq platform using the 600-cycle MiSeq reagent Kit v.3 (Illumina, USA). Sequence quality metrics were assessed using FASTQC (Andrews 2010 ).
7
Plasmid Isolation and Linearization
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An E. coli TOP10 strain harboring the plasmid pZE21 was inoculated into 10 ml LB broth with kanamycin (50 μg/ml). The culture was incubated at 37°C overnight. Plasmid isolation was performed with the GeneJET Plasmid Miniprep Kit (Thermo Fisher Scientific, Waltham, United States). The DNA concentration was measured by Qubit®; (Thermo Fisher Scientific), and the isolated plasmid was electrophoresed on a 1% agarose gel for visual inspection. Next, the plasmid was cut by HincII #R0103C (NEB, Ipswich, Massachusetts) according to manufacturer's instructions. Subsequently, the plasmid ends were dephosphorylated according to the protocol for dephosphorylation of 5′-ends of DNA by rSAP (NEB). Finally, the linearized and dephosphorylated plasmid DNA was purified with the Clean-up Concentrator kit (A&A Biotechnology, Gdynia, Poland).
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