Red fluorescent F1 progeny (n = 24 total), from successfully injected P0s, were singled to 35 mM NGM dishes and allowed to lay F2 eggs for 2 d. After egg-laying, individual F1s were placed in 7 μl of PCR lysis buffer (50 mM KCl, 10 mM Tris-HCl pH = 8.3, 2.5 mM MgCl2, 0.45% NP-40, 0.45% Tween-20) containing 1 mg/ml proteinase K in PCR strip tubes. After 1 hr at −80°, the PCR tubes were lysed for 1 hr at 60°, followed by 15 min at 95° to inactivate the proteinase K. An aliquot (4 μl) of the genomic DNA containing single worm lysate was used to PCR amplify an 820 bp fragment surrounding the targeted genomic edit using Q5 Hot Start High-Fidelity 2X Master Mix (NEB). The primer sequences for lgc-35 were: F1: 5′-TCGTCATTACGTCCTGGGTTTC-3′ and R1: 5′-CCATTGGTTCAAGACGGGTAAG-3′. PCR products were purified and eluted in 10 μl nuclease-free water using a DNA Clean and Concentrate kit (Zymo Research). The purified and concentrated PCR products were digested with ScaI-HF (NEB) for 1–2 hr and then loaded and separated on a 1.5% agarose gel.
Dna clean and concentrate kit
Manufactured by Zymo Research
Sourced in United States
The DNA Clean and Concentrate kit is a laboratory tool designed to purify and concentrate DNA samples. It facilitates the removal of contaminants and inhibitors from DNA samples, enabling the recovery of high-quality DNA for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Scai hf, by New England Biolabs (1 mentions)
Q5 hot start high fidelity 2x master mix, by New England Biolabs (1 mentions)
Nextseq 550, by Illumina (1 mentions)
Q5 dna polymerase, by Illumina (1 mentions)
Snabi, by New England Biolabs (1 mentions)
Bstbi, by New England Biolabs (1 mentions)
Dh5α competent cells, by Thermo Fisher Scientific (1 mentions)
Sacii, by New England Biolabs (1 mentions)
Gel purification kit, by Zymo Research (1 mentions)
6 protocols using dna clean and concentrate kit
1
Genotyping F2 Progeny via Restriction Digest
2
High-Throughput Sequencing Library Preparation
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Sequencing libraries were prepared as 225 bp amplicons containing the 3′ UTR libraries. Plasmids isolated before and after selection were amplified by 12–16 cycles of PCR using primers that contained Illumina adapter sequences and unique sequencing indices. PCR products were isolated using a DNA Clean and Concentrate Kit (Zymo Research) and quantified using a Qubit fluorometer. The sequencing libraries were diluted to 2 nM and denatured for sequencing following the standard Illumina protocol. DNA sequencing was performed on an Illumina Nextseq 550 instrument sequencer. To identify the set of sequences in our library, we made use of the program Bartender, which collapses similar sequences into a set of consensus sequences [67 (link)]. We ran Bartender using the following options: -t 40 -d 8 -z -1 -c 1 -l 8. This set of consensus sequences was used in all subsequent analyses, with alignments performed to these sequences using Bowtie2 [68 (link)].
3
Next-Generation Sequencing Library Preparation
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A 50 µL PCR reaction was run using 5–10% of the bead slurry products from the second ligation reaction using Q5 DNA polymerase (NEB) and following the manufacturer’s instructions: 0.02 U/µL Q5 DNA polymerase, 1x Q5 reaction buffer, 0.2 mM dNTPs, 0.5 µM Illumina index primer (Supplementary Data 5 ), and 0.5 µM Illumina multiplex primer (Supplementary Data 5 ). Typical PCR cycles were 9–15 cycles at 10 s at 98 °C, 15 s at 55 °C, and 72 °C for 20 s. PCR reactions were then processed through the DNA Clean and Concentrate kit (Zymo Research).
4
Transposon Insertion Site Identification
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MAP mutants were lysed in the deionized water with mechanical disruption method using 0.2 mL of 0.1-mm-diameter glass beads. Samples were cleared by centrifugation at 10,000× g for 5 min and DNA was purified using a DNA Clean and Concentrate kit (Zymo Research, Irvine, CA, USA) following the manufacturer’s protocol. The transposon insertion site was identified using the previously reported ligation-mediated polymerase chain reaction (LM-PCR) technique that is comprehensively described in [24 (link)]. The final PCR products were separated on the agarose gel, and purified DNA fragments sequenced at the Center for Genome Research and Biocomputing, Oregon State University.
5
Cloning and Expressing GAD65 and H3N2 HA Reactive TCRs
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The GAD65 reactive TCR expression construct was described in Ref. [29 (link)]. The sequence for the H3N2 HA reactive TCR HA1.7 was obtained from the NCBI nucleotide database: GenBank accession number X63455.1 (TCRα), and X63456.1 (TCRβ), as deposited by Hewitt et al. [30 (link)]. The variable TCRα and TCRβ chains were ordered as separate gBlocks (IDT), or with both the TCRα and TCRβ sequence in the same gBlock, containing 5′ and 3′ adaptors with respective restriction sites, and sequentially cloned into the TCR-pMSCVII-Ametrine (TCR-pMIA) plasmid [29 (link)]. Briefly, the TCRα gBlock and vector were digested with SnaBI and SacII (both New England Biolabs), and the digested gBlock product was purified with a DNA clean and concentrate kit (Zymo Research), while the digested TCR-pMIA plasmid was run on an agarose gel and the correct band purified with a gel purification kit (Zymo Research). The purified insert and plasmid backbone were ligated, and the product expanded in DH5α competent cells (Invitrogen). The insert was confirmed by sequencing, and subsequently the TCRβ was cloned into the vector by restriction digestion using MfeI and BstbI (both New England Biolabs), using the same setup as for the cloning of the TCRα chain.
6
Sequencing library preparation and analysis
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Sequencing libraries were prepared as 225 bp amplicons containing the 3′ UTR libraries. Plasmids isolated before and after selection were amplified by 12-16 cycles of PCR using primers that contained Illumina adapter sequences and unique sequencing indices. PCR products were isolated using a DNA Clean and Concentrate Kit (Zymo Research) and quantified using a Qubit fluorometer. The sequencing libraries were diluted to 2 nM and denatured for sequencing following the standard Illumina protocol. DNA sequencing was performed on an Illumina Nextseq 550 instrument sequencer. To identify the set of sequences in our library, we made use of the program Bartender, which collapses similar sequences into a set of consensus sequences [53] . We ran Bartender using the following options: -t 40 -d 8 -z -1 -c 1 -l 8. This set of consensus sequences was used in all subsequent analyses, with alignments performed to these sequences using Bowtie2 [54] .
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