PCR amplification of the lacZ gene from selected bacterial colonies generated a 547 bp amplicon using PCR Primers (Integrated DNA Technologies, Coralville, Iowa) FWD 5′-GCTTCCGGCTCGTATGTTGTGTGG-3′ and REV 5′-GTTGGACGAGTCGGAATCGCAGA-3′. The PCR conditions involved an initial denaturation of template DNA at 94 °C for 2 min, cycle denaturation at 94 °C for 30 s, primer annealing at 60 °C for 1 min, and extension at 68 °C for 30 s for 35 cycles with a hold at 68 °C for 10 min. Each PCR contained an individually picked bacterial colony, 10 µM forward and reverse primers, PCR qualified water (Quality Biological Inc., Gaithersburg, MD) and OneTaq Quick-Load Master Mix (New England Biolabs, Ipswich, MA) in a total reaction volume of 50 µl. PCR products were purified using QlAquick PCR Purification Kit (Qiagen, Hilden, Germany).
Qlaquick pcr purification kit
Manufactured by Qiagen
Sourced in Germany, Israel
The QIAquick PCR Purification Kit is a lab equipment product designed for the rapid purification of PCR amplification products. It utilizes silica-membrane technology to efficiently bind and purify DNA fragments from PCR reactions, primer-dimers, nucleotides, polymerases, and salts.
Automatically generated - may contain errors
Lab products found in correlation
Pcr primers, by Integrated DNA Technologies (2 mentions)
Pcr qualified water, by Quality Biological (2 mentions)
Dextran sulfate, by Merck Group (2 mentions)
Oligoarray complex pools, by Twist Bioscience (2 mentions)
Formamide, by Merck Group (2 mentions)
Ep7051, by Thermo Fisher Scientific (2 mentions)
Quickextract dna extraction solution 1, by Illumina (2 mentions)
Q5 high fidelity dna polymerase, by New England Biolabs (2 mentions)
Borate buffer, by Thermo Fisher Scientific (2 mentions)
Amino 11 ddutp, by Lumiprobe (2 mentions)
Biotin nhs ester h1759, by Merck Group (2 mentions)
Azidoacetic acid nhs ester 900919, by Merck Group (2 mentions)
S acetylthioglycolic acid nhs ester a9043, by Merck Group (2 mentions)
Propargyl nhs ester, by Merck Group (2 mentions)
Bio spin 30 columns, by Bio-Rad (2 mentions)
Tdt polymerase, by New England Biolabs (2 mentions)
Onetaq quick load master mix, by New England Biolabs (1 mentions)
Phusion high fidelity pcr master mix with hf buffer, by New England Biolabs (1 mentions)
3500 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
3500 data collection software, by Thermo Fisher Scientific (1 mentions)
9 protocols using qlaquick pcr purification kit
1
Amplification and Purification of lacZ Gene
2
Oligoarray-based DNA Probe Synthesis
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Primary probes were ordered as an oligoarray complex pools from Twist Bioscience and were constructed as previously described 6 (link),11 (link),21 . Briefly, a 2-step limited PCR cycles were used to amplify the designated probe sequences from the oligo complex tool. Then, the amplified products were purified using QlAquick PCR Purification Kit (28104; Qiagen) according to the manufacturer's instructions. The PCR products were used as the template for in vitro transcription (E2040S; NEB) followed by reverse transcription (EP7051; Thermo Fischer) with the forward primer. After alkaline hydrolysis, the single stranded DNA (ssDNA) probes were purified by ethanol precipitation and resuspend in primary probe hybridization buffer comprising of 30% formamide (F9037; Sigma), 2× SSC (15557036; Thermo Fischer), and 10% (w/v) Dextran Sulfate (D8906; Sigma). The probes were stored at -20°C.
3
Quantifying Gene Editing Efficiency
4
Efficient Primer and DNA Modifications
5
Oligoarray-based DNA Probe Synthesis
6
Efficient Primer and DNA Modifications
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For 5’ modification and PS bond modification: primers with 2PS bonds, Acrydite™, C6-Alex 647, amine C6 and amine C12 were ordered from IDT. Bis-PEG10-NHS ester was purchased from Broadpharm (BP-22588). Biotin NHS ester (H1759), azidoacetic acid NHS ester (900919), s-acetylthioglycolic acid NHS ester (A9043), and propargyl NHS ester (764221) were purchased from Sigma-Aldrich. NHS esters were incubated with 10 μM primers with 5’ amine C6 or C12 group at 1 mM concentration in 1 x borate buffer (Thermo Fisher, 28341) overnight at room temperature. Primers were then desalted using Bio-Spin 30 columns (Bio-rad). The labelling efficiencies were measured by HPLC and MALDI-TOF mass spectrometry (Supplementary Fig. 3 ). For 3’ Am-ddU modification: An aliquot of 10 μg dsDNA donors, 20 μM amino-11-ddUTP (Lumiprobe), 50 μM CoCl2, and 1 U TdT polymerase (New England Biolabs), 1 x TdT reaction buffer were incubated in a 50 μl reaction at 37 °C for 4 h. amino-11-ddUTP modified dsDNA was purified using QlAquick PCR purification kit (Qiagen) after stopping the reaction with 10 μl 0.2 M EDTA (PH = 8.0). The resulting product was then used to generate 3’ ddU-PEG10 modified dsDNA by incubation with Bis-PEG10-NHS ester at conditions described above.
7
Quantifying Gene Editing Efficiency
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Genomic DNA from cell pellets was extracted using QuickExtract DNA Extraction Solution 1.0 (Epicentre). Amplification of the junction regions surrounding gene KI sites in edited cells was carried out by PCR using Q5 High-Fidelity DNA polymerase (New England BioLabs) and 100 ng genomic DNA as template in a 50 μL reaction. PCR products were purified using QlAquick PCR purification kit (Qiagen) and then subjected to direct DNA sequencing service (ACGT, Wheeling, IL). The indel rates were analyzed by the online software12 (link) (http://tide.nki.nl ) using sequence without indels as reference.
8
Amplification and Sequencing of lacZ Gene
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Polymerase chain reaction (PCR) amplification of the lacZ gene from plasmid DNA isolated from selected bacterial colonies generated a 539 bp amplicon using PCR primers (Integrated DNA Technologies): fwd 5′-GCTTCCGGCTCGTATGTTGTGTGG-3′ and rev 5′-GTTGGACGAGTCGGAATCGCAGA-3′. The PCR conditions involved an initial denaturation of template DNA at 98°C for 30 s, cycle denaturation at 98°C for 10 s, primer annealing at 60°C for 30 s, and extension at 72°C for 10 s for 35 cycles, with a hold at 72°C for 10 min. Each PCR consisted of 10 ng of template DNA, 10 μM of forward and reverse primers, PCR qualified water (Quality Biological, Inc., Gaithersburg, MD), and Phusion High Fidelity PCR Master Mix with HF Buffer (New England Biolabs) in a total reaction volume of 20 μL. PCR products were purified using QlAquick PCR Purification Kit (Qiagen), and modifications were evaluated via DNA sequencing (GeneWiz, South Plainfield, NJ).
9
18S rRNA Gene Sequencing Protocol
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The PCR product of the 18S rRNA gene was purified using QlAquick PCR purification kit (QIAGEN, Germany), following the manufacturer’s instructions. Purified amplicons were sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Thermo Fisher Scientific, USA), with 1 µM primers (either 18SrRNA F or 18SrRNA R) and dideoxynucleotides (ddNTPs). The amplification products were purified using the BigDye X-Terminator purification kit (Applied Biosystems, Thermo Fisher Scientific, USA), according to the manufacturer’s protocol, and sequenced using Genetic Analyzer 3500 (Thermo Fisher Scientific, USA). Initial sequencing analysis was performed using 3500 Data Collection Software (Applied Biosystems, Thermo Fisher Scientific, USA), followed by further analysis using the basic local alignment search tool (BLAST), phymycodb, and MAFFT tools. The generated fasta sequence files were ultimately submitted to NCBI through Genbank.
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