To verify integration of the hygromycin resistance cassette into the nuclear genome of P. indica, Southern blot analysis was performed. Genomic DNA from 7-day-old cultures grown on CM medium was extracted; 10–20 μg of extracted DNA was digested overnight with SacI (NEB). The digested DNA was separated on 0.9% TAE agarose gel for 5 h at 80 V and blotted onto a nylon membrane (AmershamBiosciences Hybond-N+, GE Healthcare) over night. The DNA was UV cross-linked to the membrane in a GS GENE LINKER UV chamber (BIO-RAD) using an auto cross-linking program (C2, 50 mμ Joule). The labeling of the Hygromycin B probe was performed using the Prime-a-Gene® Labeling System according to the manufacturer’s instructions (Promega). Hybridization and washing steps were performed at 65°C. The membrane was exposed on phosphorimaging screens (Bio-Rad) and signals were detected using a molecular imager and the Quantity One software (Bio-Rad).
Phosphorimaging screens
Manufactured by Bio-Rad
Sourced in Germany
Phosphorimaging screens are reusable, radiation-sensitive plates used in conjunction with a phosphorimager to detect and analyze radioactive signals, such as those from labeled proteins, nucleic acids, or other biomolecules. They capture and store the information from radioactive samples, which can then be read and quantified using a phosphorimaging system.
Automatically generated - may contain errors
Lab products found in correlation
Prime a gene labeling system, by Promega (2 mentions)
1d quantity one software, by Bio-Rad (2 mentions)
γ 32p atp, by Hartmann Analytic (2 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Gs gene linker uv chamber, by Bio-Rad (1 mentions)
Hybond n, by GE Healthcare (1 mentions)
Hybond, by Cytiva (1 mentions)
Nylon membrane, by Carl Roth (1 mentions)
5 protocols using phosphorimaging screens
1
Genome Integration of Hygromycin Resistance in P. indica
2
Quantitative Protein-cdi-GMP Binding
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For binding assays, 1.5 μM of the appropriate protein was incubated together with 2 μl of radiolabeled c-di-GMP or GTP in a binding buffer (5 mM Tris-HCl, 12.5 mM NaCl, 2.5 mM MgCl2; and final volume of 50 μl) for 5 min at RT before 10 μl were spotted on a nitrocellulose membrane (in triplicates). After drying, the membrane was washed three times with the binding buffer, tried, and exposed on phosphorimaging screens (Bio-Rad) and analyzed with the 1D-Quantity One software (Bio-Rad).
3
Northern Blot Analysis of CRISPR RNA
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For northern blot analysis, 5 μg of total RNA were electrophoretically separated on denaturing 10% polyacrylamide gels. Following electrophoresis, RNAs were transferred to a nylon membrane (pore size 0.45 μm) via semidry electroblotting and subsequently immobilized by UV crosslinking. Radiolabeled deoxyoligonucleotides complementary to the CRISPR1-4 RNA or 5S rRNA were used as probes for signal generation via phosphor imaging. [γ-32P]-ATP (Hartmann Analytic) was used for 3′ end radiolabeling reactions with T4 polynucleotide kinase (NEB) following the manufacturer’s protocol. 5S rRNA was used for quantification. Radiolabeled probes were hybridized with membrane immobilized RNAs under low stringency conditions using the Church buffer system (Church and Gilbert, 1984 (link)). For removal of background signals, membranes were washed twice with 5x SSC buffer containing 0.01% SDS (w/v) for 5 min at 42°C. Subsequently, membranes were air-dried and exposed to phosphor imaging screens (Bio-Rad) for signal generation. For removal of hybridized probes, the membranes were incubated in 5x SSC buffer containing 0.1% SDS (w/v) for 20 min at 95°C and 80 rpm.
4
Small and mRNA Detection Protocol
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For the detection of small RNAs, 8 µg total RNA were separated on a 10% polyacrylamide gel containing 7 M urea and transferred to a nylon membrane (Roth, Karlsruhe, Germany) by semi-dry electroblotting [41 (link)]. For detection of the sRNAs, specific oligodeoxynucleotides (listed in Table S2 ) were labeled with [γ-32P]-ATP (Hartmann Analytic, Braunschweig, Germany) by a T4 polynucleotide kinase end-labeling reaction (Fermentas/Thermo Fisher Scientific, Rockford, IL, USA).
For the detection of mRNA transcripts, 10 µg total RNA were separated on a 1% agarose (w/v) formaldehyde gel and transferred to a nylon membrane (Roth, Karlsruhe, Germany) by vacuum blotting [12 (link)]. For detection of mRNA transcripts, specific PCR products (primer listed inTable S2 ) were labelled with [α-32P]-CTP (Hartmann Analytics, Braunschweig, Germany) by using the Prime-a-Gene Labeling System (Promega, Mannheim, Germany).
The membranes were hybridized overnight using the Church and Gilbert buffer system [42 (link)], washed with 0.01% SDS and 5x SSC in ddH2O and exposed on phosphorimaging screens (Bio-Rad, Feldkirchen, Germany). To analyze the intensities of the phosphorimaging signals the 1D-Quantity One software version 4.6.8 Basic (Bio-Rad, Feldkirchen, Germany) was used.
For the detection of mRNA transcripts, 10 µg total RNA were separated on a 1% agarose (w/v) formaldehyde gel and transferred to a nylon membrane (Roth, Karlsruhe, Germany) by vacuum blotting [12 (link)]. For detection of mRNA transcripts, specific PCR products (primer listed in
The membranes were hybridized overnight using the Church and Gilbert buffer system [42 (link)], washed with 0.01% SDS and 5x SSC in ddH2O and exposed on phosphorimaging screens (Bio-Rad, Feldkirchen, Germany). To analyze the intensities of the phosphorimaging signals the 1D-Quantity One software version 4.6.8 Basic (Bio-Rad, Feldkirchen, Germany) was used.
5
Northern Blot Analysis of RNA
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Total RNA was prepared using three hot phenol steps [48 (link)]. Five μg RNA were separated on 10% polyacrylamide gels containing 7 M urea. RNA was transferred to Roti-Nylon plus 0.45 μm membranes (Roth) by semi-dry electroblotting. For detection, oligodeoxynucleotides (Table A in S1 File ) were labeled with [γ-32]-ATP (Hartmann Analytik) as described in [16 (link)]. Pre-hybridization and hybridization were performed in low-stringency buffer [49 (link)] at 42°C. Membranes were washed with solutions containing 5x SSC and 0.01% SDS. Membranes were exposed to phosphorimaging screens (Bio-Rad) and analyzed with the 1D-Quantity One software (Bio-Rad).
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