Total RNA was separated either by electrophoresis on agarose/formaldehyde gels (1.3% (w/v) agarose, 1.85% (v/v) formaldehyde, 1× MOPS–EDTA–NaOAc-buffer) or on polyacrylamide (PAA)–urea gels (8% PAA, 8.3 M urea, 1× Tris–borate–EDTA buffer) and blotted onto Roti-Nylon plus membranes (Carl Roth, Germany). Hybridization of the membranes with radioactively labelled probes was carried out as described (29 (link)) with the following modifications: Incubation with washing solution I (2× SSC, 0.5% SDS) was performed at 25°C. Subsequent washes with buffer II (2× SSC, 0.5% SDS) and III (0.1× SSC, 0.1% SDS) were performed at 65°C. Signals were detected by phosphorimaging on a Typhoon FLA 9500 imaging system (GE Healthcare, USA). Oligonucleotides H01–H10 used to generate hybridisation probes are given in Supplementary Table S1 . Single-stranded RNA probes were produced by in vitro transcription of PCR fragments using the Ambion T7 polymerase maxiscript kit (Thermo Fisher Scientific, Germany) in the presence of [α-32P]-UTP (Hartmann Analytics, Germany).
Ambion t7 polymerase maxiscript kit
Manufactured by Thermo Fisher Scientific
Sourced in Germany
The Ambion T7 polymerase maxiscript kit is a reagent kit used for in vitro transcription. The kit contains T7 RNA polymerase, reaction buffer, and other necessary components for synthesizing large quantities of RNA from DNA templates.
Automatically generated - may contain errors
2 protocols using ambion t7 polymerase maxiscript kit
1
Northern Blot Analysis of RNA
2
Quantification of Synechocystis Transcripts
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RNA was isolated according to Pinto et al. 55 , separated on a denaturing 1.3% agarose gel and blotted onto Roti-Nylon plus membrane (Carl Roth, Germany). In vitro transcription of PCR fragments with the Ambion T7 polymerase maxiscript kit (Thermo Fisher Scientific, Germany) and [α-32 P]-UTP (Hartmann Analytics, Germany) was used to generate radioactively-labeled RNA probes for the 5' UTR of psbA2 mRNA and the control RNA RnpB. A PCR fragment covering the psaA sequence was labeled using the Rediprime II DNA labeling system (GE Healthcare Life Sciences).
Hybridization signals were detected on a Typhoon FLA4500 imaging system (GE Healthcare) and quantified using Quantity One software (Bio-Rad Laboratories, Germany). Primers used to amplify PCR products for labeling reactions are given in Supplementary Table 6.
Antiserum against Synechocystis Rbp3 protein.
For the expression of 6His-tagged Rbp3 protein, the PCR-amplified rbp3 gene was cloned into pETNH vector 58
Hybridization signals were detected on a Typhoon FLA4500 imaging system (GE Healthcare) and quantified using Quantity One software (Bio-Rad Laboratories, Germany). Primers used to amplify PCR products for labeling reactions are given in Supplementary Table 6.
Antiserum against Synechocystis Rbp3 protein.
For the expression of 6His-tagged Rbp3 protein, the PCR-amplified rbp3 gene was cloned into pETNH vector 58
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