Cells were fixed with formaldehyde for 10 minutes at 37°C before the reaction was stopped with 0.12M glycine and washed with PBS. Cells were resuspended and incubated on ice for 10 minutes in lysis buffer (50mM HEPES (pH 7.9), 140mM NaCl, 1mM EDTA, 1% Triton-X-100, 0.1% Na-deoxycholate) containing protease inhibitors. Lysates were then sonicated to ∼500bp and chromatin pre-cleared, followed by immunoprecipitation with antibodies and 50ul Protein A Agarose. The beads were washed as previsously described (29 (link)). DNA was reverse cross-linked and cleaned with the Illustra GFX PCR columns and gel band purification kit (GE Healthcare Cat# 28-9034-71). Samples were used for quantitative PCR on the MyIQ thermocycler (Biorad).
Illustra gfx pcr
Manufactured by GE Healthcare
Sourced in United States
The Illustra GFX PCR is a laboratory equipment product from GE Healthcare that performs polymerase chain reaction (PCR) amplification of DNA samples. It is a compact, versatile PCR system designed for a wide range of applications in life science research and diagnostics.
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2 protocols using illustra gfx pcr
1
Chromatin Immunoprecipitation and qPCR Analysis
2
Dual-indexing PCR amplicon sequencing
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After the second dual indexing PCR, the amplification products were checked using agarose gel electrophoresis across all loci and diluted appropriately to minimise the amplification rate differences between samples. Two microliters of each PCR product (across all loci and all specimens) were pooled together and cleaned using the Illustra GFX PCR and a gel band purification kit (GE Healthcare, Chicago, IL, USA), following the recommended procedures to remove shorter oligonucleotides. The cleaned sample was eluted in 25 µL, analysed with a highly accurate DNA electrophoresis Bioanalyzer 2100 system using a DNA 1000 kit (Agilent, Santa Clara, CA, USA), diluted to the final concentration of 20 ng/µL, and submitted for the Illumina 150 bp paired-end sequencing at GATC Biotech (Ebersberg, Germany). The project was designed to obtain approximately 5 M paired-end reads per DNA library. The reads were delivered as two FASTQ non-interleaved files.
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