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Caliper labchip gx

Manufactured by PerkinElmer
Sourced in United States

The Caliper LabChip GX is a microfluidic-based system designed for automated electrophoretic analysis of DNA, RNA, and protein samples. It utilizes miniaturized, integrated fluidic chips to perform rapid sample separation and detection.

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15 protocols using caliper labchip gx

1

Whole Genome Sequencing Library Prep

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Samples for WGS were processed at the Genome British Columbia Genome Sciences Centre. A PCRFree genome library construction from 500 ng of genomic DNA in 62.5 μL of elution buffer (i.e., a concentration of 8 ng/mL) was used. Different qualitative and quantitative control steps were taken for samples at various stages of the library construction (i.e., initial receipt, post-shearing, post-library construction), and these steps were as follows: QCed for quality and quantity with Caliper LabChip GX for DNA samples using the High Sensitivity Assay (PerkinElmer, Inc., USA), Agilent DNA 1000 series II assay, Quant-iT dsDNA HS Assay Kit using Qubit fluorometer (Invitrogen), and KAPA qPCR. A paired-end 125 bp sequencing was performed on the Illumina HiSeq 2500 platform, using V4 chemistry according to manufacturer recommendations. The average coverage was 367×. The overall coverage range was 232×–534×, with the exception of two samples that only got 65×, and therefore, those samples were discarded.
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2

Isolation and Quality Assessment of Total RNA

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Total RNA was isolated from CD14+ monocytes and CD14 cells using the RNeasy Mini Kit with DNase digestion on the column (Qiagen, Inc. Valencia, CA, USA) as per the manufacturer’s instructions. Total RNA was isolated from biopsy samples using the RNeasy Lipid Tissue Mini Kit with DNase digestion on the column (Qiagen, Inc. Valencia, CA, USA) as per the manufacturer’s instructions. The quality and quantity of RNA was assessed using the Agilent 2100 Bioanalyzer (South Plainfield, NJ, USA) or the Caliper LabChip GX (PerkinElmer, Inc. Waltham, MA, USA). Only those RNAs with a RNA Integrity Number (RIN, Agilent) or RNA Quality Score (RQS, PerkinElmer) of greater than 4.0 (out of 10) were submitted for microarray. CD14+ depleted PBMC and CD14+ monocyte RNAs had an average RQS of 9.4, and biopsy RNAs had an average RQS/RIN of 6.7.
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3

Bacterial RNA Enrichment and Sequencing

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Total RNA was isolated from infected plant samples using the EZNA Plant kit (Omega) according to the manufacturer's recommendations, including the on‐column DNase treatment, which was carried out using Turbo DNase I (Invitrogen). RNA was checked for quality and quantity using Caliper LabChip GX (Perkin Elmer) and Qubit RNA HS (Thermo Fisher Scientific) analysis, respectively. To enrich RNA samples for bacterial messenger RNA, multiple depletion reagents were used, including oligo(dT) beads to deplete eukaryotic polyadenylated mRNA, RiboZero Plant rRNA removal beads to deplete host rRNA, and RiboZero bacterial rRNA removal beads to remove prokaryotic rRNA (Illumina). The remaining RNA was used for library preparation using an Illumina TruSeq Stranded Total RNA Library Preparation Kit (Illumina) on a Sciclone G3 robot following the manufacturer's recommendations (Perkin Elmer). Library quality was assessed using Caliper LabChipGX HS DNA (Perkin Elmer) and Qubit dsDNA HS (Thermo Fisher Scientific) assays. Samples were pooled and run on three lanes using the Illumina HiSeq4000 platform with single‐end 50 bp format. Sequencing was conducted at the Michigan State University Research Technology Support Facility. Base calling was conducted by Illumina Real Time Analysis v. 2.7.6.
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4

Illumina Library Preparation and Sequencing

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Illumina library preparation, hybridization capture, and sequencing was conducted at the Yale Center for Genomic Analysis (YCGA). Library preparation was conducted using a modified Roche/Nimblegen SeqCap EZ Library Short Read protocol [68 ]. Library concentration was determined using PicoGreen assay (Invitrogen) and size selection was performed on a Caliper LabChip GX instrument (PerkinElmer).
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5

Quantitative Analysis of Notch Pathway Genes

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Total RNAs were isolated using Trizol reagent (Life Technologies), phenol-chloroform extraction and isopropanol precipitation. RNAs were analyzed using a microchip capillary electrophoresis system (Caliper LabChip GX, PerkinElmer, Waltham, MA, USA). RNA with RQS >7 (RNA Quality Score) were treated with ribonuclease-free Turbo-DNase (Life Technologies) before reverse transcription (RT). RTs were performed with Moloney murine leukemia virus reverse transcriptase (Life Technologies) according to the manufacturer’s instructions. Real-time quantitative PCR was performed with a ViiA™ 7 sequence detection application program using the following labeled ready-to-use primers/probe mixes (Life Technologies): Notch1 (Hs01062014_m1), Notch2 (Hs01050702_m1), Notch3 (Hs01128541_m1), Notch4 (Hs00270200_m1), DLL1 (Hs00194509_m1), DLL3 (Hs00213561_m1), DLL4 (Hs00184092_m1), Jag1 (Hs01070036_m1), Jag2 (Hs00171432_m1), HES1 (Hs00172878_m1), HEY1 (Hs01114113_m1), IRF5 (Hs00158114_m1), STAT1 (Hs01013996_m1), SOCS3 (Hs02330328_s1), TNF (Hs01113624_g1), IL-6 (Hs00174131_m1), IL-1β (Hs0155410_m1), CD40 (Hs00334176_m1), c-myc (Hs00811069_g1). QPCR for β actin (Actb) (Hs99999903_m1) was used as an endogenous control and for data normalization. Relative gene expression was calculated according to the 2−ΔΔCt method.
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6

Genotyping Rat Aire Gene Mutations

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Small ear biopsies were taken from 10-day-old rats to perform genotyping as previously described (51 (link)). Briefly, samples were digested overnight at 56°C in 350 μL of lysis buffer, then diluted at 1:20 in ultrapure water and added to 24 μL of PCR mix reaction prepared according to the manufacturer protocol (Herculase II Fusion DNA Polymerase, Agilent Technologies). The following primers were used to carry out the PCR amplification: forward primer 5′-TCAAGAGTGCCCTGTTCTAG-3′, reverse primer 5′-CTGGGGTGGTGTCAGTAAG-3′. The amplification program was run on a Veriti Thermal Cycler (Applied Biosystems) and consisted of 1 cycle at 95°C for 5 minutes, 35 cycles at 98°C for 10 seconds, 60°C for 10 seconds, and 72°C for 30 seconds, followed by 1 cycle at 72°C for 4 minutes. Finally, to determine the presence of mutations in the Aire gene, the PCR product’s length was estimated using a heteroduplex mobility assay with a microfluidic capillary electrophoresis system (Caliper LabChip GX, PerkinElmer).
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7

Transcriptomic Analysis of Striatal Subregions

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Naive S4 mice (N = 12/line/sex) were euthanized, brains removed and immediately frozen on dry ice. Frozen brains were sliced in 55-μ coronal sections on a freezing microtome at −13°C and slices containing the NAc were mounted on polyethylene naphthalate-covered slides. Mounted slices were lightly thionin stained under RNAse-free conditions and dehydrated in increasing concentrations of ethanol diluted in RNAse-free water (50%, 70%, 95% and 100%) for 30 seconds each and then air-dried. The NAc shell was dissected bilaterally on a Leica LMD-6000 (Leica Microsystems Inc., Buffalo Grove, IL) using known anatomical landmarks (Paxinos & Franklin 2007 ). Dissected tissue was processed with the Arcturus Picopure Kit (ThermoFisher Scientific, Waltham, MA) yielding on average 200 ng of total RNA. RNA quality was assessed using the Caliper Labchip GX (PerkinElmer, Waltham, MA) and RNA Quality Scores (RQS).
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8

RNA Extraction from FFPE Specimens

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RNA extracted from FFPE specimens was highly fragmented as is typical of this sample type. The RNA Quality Scores (RQS) from Caliper LabChip GX (Perkin-Elmer, Waltham, MA) HT RNA assays ranged from 1.8 to 6.5 with an average of 3.0 for Set 1 samples and from 1.7 to 7.4 with an average of 2.7 for Set 2. In both sample groups the majority of the samples had an RQS between 2 and 3 with RNA fragment sizes distributed between 25 and approximately 500 bases.
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9

Long-range Whole Genome Sequencing

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Prior to 10X Genomics lrWGS library construction, genomic DNA samples were checked for fragment size distribution and were quantified. Genomic DNA fragment size distributions were determined with a Caliper Lab Chip GX (Perkin Elmer) to quantify DNA above 40 kb in length. Size selection was performed on 1.2 ug of genomic DNA with an 0.75% Agarose cassette on the Blue Pippin platform (Sage Science) with target specifications set to start at 40 kb and end at 80 kb. Samples were quantified using the Quant-it Picogreen assay kit (Thermo Fisher) on a Qubit 2.0 Fluorometer (Thermo Fisher) and normalized to a starting concentration of 1 ng/uL with TE (0.1 mM EDTA). Starting concentrations of 1 ng/uL were confirmed by picogreen and libraries were subsequently created in accordance with the 10X WGX protocol (10X Genomics). Library size was determined using the DNA 1000 Kit and 2100 BioAnalyzer (Agilent Technologies) and quantified using quantitative PCR (qPCR) (KAPA Library Quantification Kit, Kapa Biosystems). The finished WGX libraries were run on an Illumina HiSeqX platform at paired 151 bp reads with an eight-base single index read at the Broad Institute. Upon completion of sequencing, the resulting BCL files were processed by the Long Ranger Pipeline (10X Genomics) for alignment, variant discovery, and phasing.
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10

Nextera XT DNA Library Preparation

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DNA libraries were prepared with Nextera XT DNA Library Preparation Kit (Illumina, California, USA). Quality control was performed with Caliper LabChip GX (Perkin Elmer) prior to shotgun sequencing with MiSeq (Illumina, California, USA), with an expected sequencing depth of 260 Mb/library (expected coverage > 80×). One hundred twenty-nine million reads were generated (704 thousand reads/sample s.d. 349 thousand).
Sequences were pre-processed by removing low-quality (mean quality lower than 25) or low-complexity reads, reads mapping to human genome or to large and small ribosomal units of bacteria, fungi and human, and known contaminants (e.g. phiX174, Illumina spike-in). All genomes are available at the NCBI Sequence Read Archive (BioProject accession number PRJNA400143).
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