Human IL-10 was amplified from pUbC-hIL-10 (provided by Prof. David Dean, University of Rochester Medical Center) and ligated into the viral plasmid backbone, HIV-CSCG (provided by Prof. David Schaffer, University of California, Berkeley) using NheI and XhoI (New England Biosciences, Ipswich, MA). Lentivirus reporter constructs containing enhancer elements bound by NF-κB and encoding firefly luciferase were employed to assay transcription factor (TF) activity along with a negative control construct containing only a TATA box promoter (Panomics, Freemont, CA) by live imaging (Weiss et al., 2012 (link)). Ligated plasmids were transformed into DH5α cells (Invitrogen). Plasmids were purified using Qiagen reagents and stored in Tris–EDTA buffer (Qiagen, Venlo, Limburg, Netherlands) at −20°C.
Tris edta buffer
Manufactured by Qiagen
Sourced in Netherlands, United States
Tris-EDTA (TE) buffer is a commonly used buffer solution in molecular biology and biochemistry. It is a mixture of Tris-HCl and EDTA, which serves to maintain the pH and chelate divalent cations, respectively. TE buffer is primarily used for the storage and dilution of nucleic acids, such as DNA and RNA, to help preserve their integrity and stability.
Automatically generated - may contain errors
Lab products found in correlation
Ez1 dna tissue kit, by Qiagen (3 mentions)
Proteinase k, by Qiagen (3 mentions)
G2 lysis buffer, by Qiagen (3 mentions)
Dh5α cells, by Thermo Fisher Scientific (1 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (1 mentions)
Hplc purified oligos, by Integrated DNA Technologies (1 mentions)
Nanodrop nd 1000 uv vis spectrophotometer, by Thermo Fisher Scientific (1 mentions)
6 protocols using tris edta buffer
1
Generation of Lentiviral Constructs for NF-κB Assay
2
Genomic DNA Extraction from Snails
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The remaining body of each specimen was used for genomic DNA extraction as a substrate to detect the parasite and confirm the molecular identity of the snails. DNA was extracted using the EZ1 DNA Tissue kit (Qiagen) following the manufacturer’s recommendations. Each snail specimen was placed in a 1.5 mL Eppendorf tube and incubated at 56°C overnight in 180 μl of G2 lysis buffer (Qiagen Hilden, Germany) and 20 μl of proteinase K (Qiagen Hilden, Germany). The supernatant was recovered in another tube and then extracted using the EZ1 BioRobot extraction device (Qiagen Hilden, Germany). Genomic DNA from each sample was eluted with 200 μl of Tris-EDTA buffer (Qiagen) and stored at -20°C until use.
3
Bed Bug DNA Extraction Protocol
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Bed bug DNA was extracted from the halves of each bed bug after overnight incubation at 56 °C in a 1.5 ml tube containing 180 µl of G2 lysis buffer and 20 µl of proteinase K (Qiagen, Hilden, Germany). The EZ1 DNA tissue kit (Qiagen) was used for DNA extraction in accordance with the instructions provided by the manufacturer. DNA from each sample was eluted with 100 µ of Tris–EDTA (TE) buffer (Qiagen), observed using Thermo Scientific NanoDrop 1000 (United States), and kept at − 20 °C until further analysis25 (link).
4
Tick DNA Extraction Protocol
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Each half-tick without legs was transferred to a 1.5 mL tube containing 180 μL of G2 lysis buffer and 20 μL proteinase K (Qiagen, Hilden, Germany), and incubated at 56°C overnight. DNA extraction from the half-tick was performed with an EZ1 DNA Tissue Kit (Qiagen) according to manufacturer recommendations. The DNA from each sample was eluted with 100 μL of Tris-EDTA (TE) buffer (Qiagen) and was either immediately used or stored at -20°C until use.
5
CRISPR-Cas9 Editing of TMEM67 Gene
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GFP-expressing pSpCas9(BB)-2A-GFP (PX458) (gift from Feng Zhang, Addgene plasmid #48138). Three crRNAs targeting human TMEM67 (RefSeq NM_153704.5) were designed using Benchling ( https://benchling.com ), selected for the highest ranking on-and off-target effects. crRNAs were ordered as HPLC-purified oligos from Integrated DNA Technologies in addition to Alt-R CRISPR-Cas9 tracrRNA-ATTO550 conjugates. crRNA sequences are listed in Table S6. Lyophilised pellets were resuspended in Tris-EDTA (TE) buffer (Qiagen) to give 100mM stocks. crRNA and tracrRNA were mixed GFP positive cells were present, the top 5% were targeted for index sorting. After sorting, cells were incubated for 3 weeks at 37°C with 5% CO2, with weekly checks for growing colonies.
6
Microneedle-Mediated Plasmid DNA Delivery
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MNs were visualized under the stereomicroscope throughout the coating process. A 4.5 µl (Tris-EDTA (TE) buffer, Qiagen) solution of a plasmid DNA 60 µg cocktail containing 15 µg of each recombinant plasmid from L. infantum (pcDNA3-LiH2A, pcDNA3-LiH2B, pcDNA3-LiH3 and pcDNA3-LiH4; pHIS) was loaded into a 10 µl ultra-long tip using a 2.5 µl pipette. The plasmid DNA cocktail was then deposited onto three rows of ten MN arrays, using a brushing method, reported previously (Zhao et al., 2016) , resulting in a total coating of 60 µg across 30 individual MNs (optimization studies, as shown in Fig. 1f, investigated application of a target dose of 10, 20, 30 or 60 µg of DNA per 30 MNs). The same procedure was also used to coat 60 µg of nonrecombinant plasmid (4.8 µl, as negative control; pC). To determine if the coating process affected plasmid stability, the MN coated material was recovered in 150 µL of TE buffer.
Aliquots from the TE buffer were then loaded into a 2% agarose gel supplemented with ethidium bromide and was subject to electrophoresis at 100 V for 1 h. Coating efficiency was calculated by recovering the coated pDNA from the MNs, in 200 µl TE buffer, for quantitative analysis (NanoDrop®, ND-1000 UV-Vis Spectrophotometer; NanoDrop Technologies).
Aliquots from the TE buffer were then loaded into a 2% agarose gel supplemented with ethidium bromide and was subject to electrophoresis at 100 V for 1 h. Coating efficiency was calculated by recovering the coated pDNA from the MNs, in 200 µl TE buffer, for quantitative analysis (NanoDrop®, ND-1000 UV-Vis Spectrophotometer; NanoDrop Technologies).
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