Strains carrying plasmids with GAL::RECQL5, GAL::RECQL5-ID, or the empty vector (pYES) were grown selective in SC medium with 2% raffinose to mid-log phase and then 2% galactose was added to induce transcription from the GAL1 promoter. Samples were taken at different times and RNA extraction was performed by acid phenol following standard procedures. Northern blot of kinetics was carried out as previously described (Chávez and Aguilera 1997 (link)). RNA was separated by agarose electrophoresis in a gel containing MOPS 1 × and 2% formaldehyde and transferred to Hybond-N nitrocellulose membranes (GE Healthcare). GAL1 and GAL10 probes were amplified by PCR using primers detailed in Supplementary Table 1. Probes were 32P-labelled using Klenow polymerase (Roche) and 32P-dCTP. Signal was acquired using a FLA-5100 Imager Fluorescence Analyzer (Fujifilm) and quantified with MultiGauge 2.0 analysis software (Science Lab). Signal was measured as arbitrarily units (a.u.) and normalized to control condition.
Klenow polymerase
Manufactured by Roche
Klenow polymerase is a DNA-dependent DNA polymerase enzyme derived from the Escherichia coli DNA polymerase I. It is commonly used in molecular biology applications for DNA synthesis and manipulation.
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3 protocols using klenow polymerase
1
Kinetics of GAL gene expression
2
DNA-Protein Binding Assay for LRH-1 Response Element
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Double-stranded oligonucleotides of the following sequences were used as probes: LRH-1 response element (LRE) in the human SHP promoter (LRE WT (-518 to -489), 5'-GAAGCA-GGGCCCAAGGTTAGGCAACAAG-3', LRE is underlined); LRE mutant competitor (LRE MT, 5'-GAAGCAGGGCCCttc-caaAGCAACAAG-3'); consensus LRE (5'-GACTTCTGGAGTCAAGGTTGTTGGGCCATTC-3'). The probes were labeled with [α-32P] dATP using Klenow polymerase (Roche, Mannheim, Germany), and the labeled probe (35,000 cpm) was incubated with nuclear extracts of HepG2 cells treated with 100 nmol/L T3 in 10 mmol/L Hepes (pH 7.9) containing 50 mmol/L KCl, 0.1 mmol/L ethylenediaminetetraacetic acid, 0.25 mmol/L DTT, 0.1 mg/mL poly (dIdC), 0.01% nonidet P-40, and 10% glycerol at room temperature for 10 minutes. Competitors were added in 50-fold molar excess to the labeled probe.
3
Northern Blot Analysis of RNA
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RNA was separated by agarose electrophoresis in a gel containing MOPS 1× and 2% formaldehyde and transferred to Hybond-N nitrocellulose membranes (GE Healthcare). LYS2 and SCR1 probes were amplified by PCR using primers ‘LYS2 probe A fw’ and ‘rv’ or ‘SCR1 .483 rv’ and ‘SCR1 .99 dw’, respectively, purified using Macherey-Nagel's DNA extraction kit and 32P-labelled using Klenow polymerase (Roche) and 32P-dCTP. Signal was acquired using a FLA-5100 Imager Fluorescence Analyzer (Fujifilm) and quantified with MultiGauge 2.0 analysis software (Science Lab). Signal was measured as arbitrarily units (a.u.) and normalized to control condition.
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