Standard protocols were followed to isolate the genomic DNA from rice leaves and to perform Southern blot hybridization [27 ]. In brief, the HindIII-digested genomic DNA (12 μg) upon electrophoresis on agarose gel overnight, was transferred onto nylon membrane (Hybond-N+). A 470 bp DNA fragment from the middle of SAPK9 coding DNA sequence (CDS) from O. rufipogon was PCR-amplified using gene-specific primers SA9SF-SA9SR (Additional file 1 : Table S1) and radiolabelled with P32-dCTP (3500 Ci/mmol) by random priming using rediprime II DNA labelling system (GE Healthcare, USA) following the vendor’s instructions. For autoradiography, the Cylone® Plus phosphor system (Perkin Elmer) was used to scan the multi-sensitive X-ray film (Perkin Elmer).
Rediprime 2 dna labelling system
Manufactured by GE Healthcare
Sourced in United States
The Rediprime II DNA Labelling System is a tool for the random prime labelling of DNA. It provides a reliable and efficient method for the incorporation of labeled nucleotides into DNA samples, which can then be used for various applications such as probe generation and hybridization studies.
Automatically generated - may contain errors
Lab products found in correlation
Hybond, by Cytiva (2 mentions)
Cylone plus phosphor system, by PerkinElmer (1 mentions)
Dneasy kit, by Qiagen (1 mentions)
Hybond n, by GE Healthcare (1 mentions)
Rapid hyb buffer, by GE Healthcare (1 mentions)
Positively charged nylon membrane, by Roche (1 mentions)
Ultrahyb ultrasensitive hybridization buffer, by Thermo Fisher Scientific (1 mentions)
Amersham typhoon 5 biomolecular imager, by GE Healthcare (1 mentions)
Amersham typhoon, by Cytiva (1 mentions)
3 protocols using rediprime 2 dna labelling system
1
Southern Blot Analysis of Rice SAPK9 Gene
2
DNA Blotting and Radioactive Probing
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Genomic DNA was extracted from the cells using a DNeasy kit (Qiagen). Ten micrograms of the DNA were electrophoresed and transferred to nylon membranes (Hybond-N+, GE Healthcare, Fairfield, CT). The DNA was then crosslinked to the membrane by ultraviolet light. The EGFP probe was prepared from MIGR1 vector by cutting it using NcoI and SalI, and was labeled with 32P-dCTP using the Rediprime II DNA Labelling System (GE Healthcare). The membrane was hybridized with the probe in Rapid-hyb buffer (GE Healthcare), and was analyzed by a Phosphoimager (LAS1000, Fuji Film, Tokyo, Japan).
3
Northern Blot Analysis of MERS-CoV RNA
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Total RNA extracted from the infected cells was denatured in a denaturing RNA loading buffer by incubation at 65°C for 5 min prior to gel electrophoresis on a 0.7% denaturing agarose gel. Following visualization of RNAs by ethidium bromide (EtBr) staining, the separated RNAs were transferred to a positively charged nylon membrane (Roche) via capillary action overnight and crosslinked by UV light. The membrane was then prehybridized using ULTRAhyb Ultrasensitive Hybridization Buffer (Thermo Fisher Scientific). DNA probe corresponding to the MERS-CoV genome ranging from nt 29,161–30,055 was radiolabeled with [α-32P]dCTP using the Rediprime II DNA labelling system (GE Healthcare Life Sciences). Hybridization of the DNA probe was carried out at 42°C in a hybridization buffer for 24 h. Membranes were washed and subjected to image analysis using an Amersham Typhoon 5 Biomolecular Imager (GE Healthcare Life Sciences).
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