Total RNA (10 μg for 10- and 20-year-old rhesus macaque brain samples, 2 μg for fetal macaque hippocampal primary neurons) was denatured using NorthernMax®-Gly sample loading dye (Ambion) and resolved on 1.2% agarose gel in MOPS buffer. The gel was soaked in 1 × TBE for 20 min and transferred to a Hybond-N + membrane (GE Healthcare) for 1 h (15 V) using a semi-dry blotting system (Bio-Rad). Membranes were dried and UV-crosslinked with 150 mJ/ cm2 at 254 nm. Pre-hybridization was done at 68 °C for 1 h, and using DIG Northern Blot Starter KIT (Roche) DIG-labeled in vitro transcribed circCACNA2D1, circCACNA1E, CDRA1s, and circMbl junction site-targeting probes were hybridized overnight. The membranes were washed three times in 2 × SSC, 0.1% SDS at 68 °C for 30 min, followed by three 30 min washes in 0.2 × SSC, 0.1% SDS at 68 °C. The immunodetection was performed with anti-DIG AP-conjugated antibodies. Immunoreactive bands were visualized using CDP star reagent (Roche) and a LAS-4000 detection system (GE Healthcare).
Dig northern blot starter kit
Manufactured by Roche
Sourced in Germany
The DIG Northern Blot Starter KIT is a laboratory tool used for the detection and analysis of RNA molecules. It provides the necessary reagents and components to perform the northern blotting technique, which allows for the size-separation, transfer, and detection of RNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Hybond n membrane, by GE Healthcare (3 mentions)
Cdp star reagent, by Roche (3 mentions)
Las 4000 detection system, by GE Healthcare (3 mentions)
Northernmax gly sample loading dye, by Thermo Fisher Scientific (3 mentions)
Semi dry blotting system, by Bio-Rad (3 mentions)
Hybond, by Cytiva (3 mentions)
Las 4000, by Cytiva (3 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Merck Group (1 mentions)
Riboruler high range rna ladder, by Thermo Fisher Scientific (1 mentions)
Dig rna labeling kit, by Roche (1 mentions)
5 protocols using dig northern blot starter kit
1
Quantification of Circular RNAs in Primate Brain
2
Isolation and Analysis of Bacterial RNA
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Total RNA was isolated from S. Typhimurium 14028 and derivatives as follows: at appropriate time points, culture samples were taken and resuspended in TRIzol reagent (Sigma-Aldrich, Taufkirchen, Germany). RNA was then isolated as previously described29 (link) and treated with DNaseI (Fermentas) twice to eliminate any DNA contamination. Synthesis of cDNA and qRT-PCR were performed as previously described77 (link). Northern blotting was performed according to Kröger and colleagues29 (link) using the DIG Northern blot starter kit (Roche, Penzberg, Germany) following the manufacturer’s manual; the RiboRuler High Range RNA ladder (Thermo Fisher, Waltham, MA, USA) was used as a marker. The oligonucleotides used for the amplification of non-radiolabeled riboprobes are listed in Table S3 .
3
Northern blot analysis of hrrF and 5S rRNA
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Northern blots were performed using the DIG Northern Blot Starter Kit (Roche, Indianapolis, IN). The RNA probes were generated using T7 RNA polymerase from the DIG RNA Labeling Kit (Roche, Indianapolis, IN) and the DNA template generated from primers ES173 and ES174 (hrrF) or ES89 and ES90 (5S rRNA). Northern blots were developed using CDP-Star ready-to-use solution (Roche, Indianapolis, IN).
4
Northern Blot Analysis of Gria1 in Macaque Brain
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Total RNA (10 μg for 10- and 20-year old male macaque brain samples, 2 μg for fetal macaque hippocampal primary neurons) was denatured using NorthernMax®-Gly sample loading dye (Ambion) and resolved on 1.2% agarose gel in MOPS buffer. The gel was soaked in 1×TBE for 20 min and transferred to a Hybond-N+ membrane (GE Healthcare) for 1 h (15 V) using a semi-dry blotting system (Bio-Rad). Membranes were dried and UV-crosslinked with 150 mJ/cm2 at 254 nm. Pre-hybridization was done at 68 °C for 1 h, and using the DIG Northern Blot Starter Kit (12039672910, Roche). DIG-labeled in vitro transcribed Gria1 junction site-targeting circular, and junction site-nontargeting linear probes were hybridized overnight. The membranes were washed three times in 2 × SSC, 0.1% SDS at 68 °C for 30 min, followed by three 30 min washes in 0.2 × SSC, 0.1% SDS at 68 °C. The immunodetection was performed with anti-DIG AP-conjugated antibodies. Immunoreactive bands were visualized using CDP star reagent (Roche) and a LAS-4000 detection system (GE Healthcare). The primer sequences of probes were seen in Supplementary Data 3 .
5
Northern Blotting of lncMtDLoop in AD
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Total RNA (10 μg for human postmortem PFC and hippocampal frozen tissues of AD and control individuals as well as wild type and 3xTg mouse brain fresh tissues, 2 μg for mouse primary neurons) was denatured using NorthernMax ® -Gly sample loading dye (Ambion) and resolved on 1.2% agarose gel in MOPS buffer. The gel was soaked in 1×TBE for 20 min and transferred to a Hybond-N+ membrane (GE Healthcare) for 1 h (15 V) using a semi-dry blotting system (Bio-Rad). Membranes were dried and UVcrosslinked with 150 mJ/ cm 2 at 254 nm. Pre-hybridization was done at 68 °C for 1 h, and using DIG Northern Blot Starter KIT (12039672910, Roche). DIG-labeled in vitro transcribed lncMtDLoop, and control probes were hybridized overnight. The membranes were washed three times in 2× SSC, 0.1% SDS at 68 °C for 30 min, followed by three 30 min washes in 0.2× SSC, 0.1% SDS at 68 °C. The immunodetection was performed with anti-DIG AP-conjugated antibodies. Immunoreactive bands were visualized using CDP star reagent (Roche) and a LAS-4000 detection system (GE Healthcare). The primer sequences of probes were seen in the Supplementary Table 2.
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