The human NANOG promoter was amplified from pNanog-Luc (Addgene #25900) using the forward primer hNan-F2 (or -F1) and the reverse primer hNan-R1 (or -R2); the primers in parentheses are fluorescently labeled versions of the listed primers; see Supplementary Table 2 . The 404 bp PCR fragments were purified using QIAquik Gel Extraction Kit (Qiagen). The pGL-NanogP-5E minus plasmid53 (link) containing the mouse 1535 bp NANOG promoter and 1337 bp enhancer (−5 kbp from NANOG promoter) was digested with PvuI (NEB) to linearize the plasmid. The DNA fragment containing NANOG promoter and enhancer was purified using QIAquik Gel Extraction Kit (Qiagen).
Qiaquik gel extraction kit
Manufactured by Qiagen
Sourced in India
The QIAquik Gel Extraction Kit is a laboratory equipment product designed for the purification of DNA fragments from agarose gels. It provides a simple and efficient method for extracting and purifying DNA from gel slices.
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Lab products found in correlation
Mutation surveyor software version 4, by SoftGenetics (1 mentions)
Abi prism 3730xl genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Pnanog luc, by Addgene (1 mentions)
Qiaamp dna micro kit, by Qiagen (1 mentions)
α select gold efficiency chemically competent cells, by Meridian Bioscience (1 mentions)
Phusion high fidelity dna polymerase, by Thermo Fisher Scientific (1 mentions)
Veriti 96 well thermal cycler, by Thermo Fisher Scientific (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Xhoi, by New England Biolabs (1 mentions)
Snapgene software, by GSL Biotech (1 mentions)
Quick dna fungal bacterial miniprep kit, by Zymo Research (1 mentions)
Wizard plus sv minipreps dna purification system, by Promega (1 mentions)
E coli jm109 competent cells, by Promega (1 mentions)
Pgem t, by Promega (1 mentions)
6 protocols using qiaquik gel extraction kit
1
Amplifying the human NANOG promoter
2
Sanger Sequencing Validation of Genetic Variants
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Sanger sequencing was performed to confirm the results identified in the patients using whole‐exome sequencing. The primers (Table S1 ) for CLCN7 (NM_001287.6) and TCIRG1 (NM_006019.4) were designed by UCSC ExonPrimer online software (http://genome.ucsc.edu/index.html ). The target sequences were amplified using polymerase chain reaction (PCR) and examined by electrophoresis in a 1% agarose gel. The products were purified using a QIAquik Gel Extraction Kit (Qiagen), and the sequencing was performed on an ABI Prism 3730XL Genetic Analyzer (Applied Biosystems; Thermo Fisher). The sequence data obtained were analyzed with Mutation Surveyor® software, version 4.0.4 (SoftGenetics).
3
Molecular Identification of Hummingbird Mites
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Mites from Anna’s Hummingbirds were removed from feathers stored in ethanol and placed into open, individual Eppendorf tubes and allowed to dry overnight at 25°C. DNA was extracted from individual mites using a commercial kit (Qiagen QIAamp DNA Micro Kit: Qiagen Inc., Valencia, CA). An additional pre-incubation step was added to the manufacturer’s protocol in which mites were incubated overnight at 56°C in 180μL Buffer ATL in an orbital shaker at 26.2 rad/s. A 644-bp region of the cytochrome C oxidase I (COI) gene was amplified by PCR using primers bcdF05 and bcdR04 [19 ]. Amplification conditions were modified slightly from the original reference: a reaction volume of 50μL was used and the number of amplification steps was increased from 40 to 45. DNA bands were extracted using a commercial kit (QIAquik Gel Extraction Kit: Qiagen Inc., Valencia, CA) according to manufacturer’s instructions. DNA was sequenced at a University of California, Davis laboratory (fDNA Sequencing Facility, Davis, CA) using the forward primer. Sequences were archived with GenBank (Accession #s MF802486 and MF802487).
4
Generating p62 Deletion Mutants
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Deletions of p62 were produced through PCR, using the pEGFP-p62 wild type as a template and specific primers generated by SnapGene software (GSL Biotech) (Supplementary Table 1 ). The generated fragments were then ligated into the pEGFP-C2 vector. Forward primers included a BglII restriction site. PCRs were performed using Phusion High-Fidelity DNA polymerase (Thermo Scientific) following the PCR standard protocol. PCR reactions were run on a Veriti 96-Well Thermal Cycler (Applied Biosystems). p62 fragments were then digested with BglII (sticky end, NEB) and run on agarose gel which was then purified. The pEGFP-C2 vector was digested with SmaI (blunt end, NEB) and run on agarose gel followed by purification with QIAquik GEL Extraction Kit (Qiagen). The pEGFP-C2 vector was then digested with BglII, followed by gel purification. After double digestion, the vector was dephosphorylated by calf intestinal alkaline phosphatase (Fermentas). The digested p62 fragments were ligated into the double-digested pEGFP-C2 vector (sticky end/blunt-end ligation) using the T4 DNA Ligase (New England BioLabs). Bacterial transformation was performed as described above using α-select GOLD Efficiency chemically competent cells (Bioline). FLAG-p62 and FLAG-C105AC113Ap62 for lentiviral expression were subcloned into the pLENTI6/V5-DEST vector using EcoRI and XhoI (NEB) as described above.
5
Bacterial 16S rDNA Sequencing Protocol
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For the genetic characterization of bacteria, 16S rDNA sequencing was performed as described earlier by [25, 26] (link). DNA extraction and purification were done by Quick-DNA Fungal/bacterial Miniprep Kit (Zymo Research, India). By using universal primers 27F (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1492R (5′GGT TAC CTT GTT ACG ACT T-3′), the 16S rDNA gene was amplified and, purification of amplicons was done by using QIAquik Gel extraction kit (Qiagen, India). After DNA sequencing of amplicons, the results were compared against GenBank database using the basic local alignment search tools (BLAST). The sequence of respective strains was deposited in the National Centre for Biotechnology Information (NCBI) database (http://www.ncbi.nlm.nih.gov/BLAST). The sequences thus obtained were submitted to the GenBank for their accession numbers. The evolutionary relationship of sequences was determined. The Phylogenetic tree was made using maximum likelihood from PhyML integrated into Unipro UGENE.
6
GABA Cl subunit HG1 genetic diversity
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The genetic diversity of the GABA Cl subunit HG1 locus (Blackhall et al., 2003) , as well as that of GluCl α and β subunit loci (Blackhall et al., 1998) (Supplementary Fig. S2A) was examined by single strand conformation polymorphism (SSCP) using previously described PCR primers (Blackhall et al., 2003) . The thermal cycler conditions used were: 95 o C for 4 minutes; followed by 40 cycles of 95 o C for 30 seconds, 54 o C for 30 seconds, and 72 o C for 30 seconds; a final extension stage of 72 o C for 5 minutes. Amplicons were first visualised on a 1.2% agarose gels and then run on non-denaturing polyacrylamide gels as previously described (Skuce et al., 2010) .
To confirm the allelic assignments of the GABA Cl subunit HG1 SSCP genotyping, the PCR products produced from two individual MHco3.N1.F2 L3 DNA heterozygotes (6F and 9G) were cloned and sequenced. Briefly, amplicons were run on a 1% agarose electrophoretic gel to enable the excision of 305 bp bands from which DNA was then isolated (QIAquik Gel Extraction kit, Qiagen).
DNA was ligated into pGEM -T (Promega) plasmid vectors to allow transformation into JM109 competent E. coli cells (Stratagene). Plasmid DNA from the cultured transformed cells was then purified using a Wizard Plus SV Minipreps DNA Purification System (Promega) and sequenced using SP6 and T7 universal primers in both orientations.
To confirm the allelic assignments of the GABA Cl subunit HG1 SSCP genotyping, the PCR products produced from two individual MHco3.N1.F2 L3 DNA heterozygotes (6F and 9G) were cloned and sequenced. Briefly, amplicons were run on a 1% agarose electrophoretic gel to enable the excision of 305 bp bands from which DNA was then isolated (QIAquik Gel Extraction kit, Qiagen).
DNA was ligated into pGEM -T (Promega) plasmid vectors to allow transformation into JM109 competent E. coli cells (Stratagene). Plasmid DNA from the cultured transformed cells was then purified using a Wizard Plus SV Minipreps DNA Purification System (Promega) and sequenced using SP6 and T7 universal primers in both orientations.
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