After IVT, RNA was purified using MEGAclear™ Transcription Clean-up Kit (ThermoFisher, UK) according to the manufacturer's protocol. In order to assess the quality of the RNA, purified RNAs and the RNA Millennium Marker Ladder (ThermoFisher, UK) were mixed with 2Â RNA loading dye (ThermoFisher, UK) and incubated at 50 °C for 30 min to denature the RNA. A 1.2% agarose gel with 1Â NorthernMax Running Buffer (ThermoFisher, UK) was prepared. After incubation, the denatured ladder and samples were added to the gel and the gel was run at 80 V for 45 min. The gel was then imaged on a GelDoc-It2 (UVP, UK).
Northernmax running buffer
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
NorthernMax Running Buffer is a buffer solution designed for use in northern blot analysis. It is used to create an optimal environment for the separation and transfer of RNA samples during the northern blot process.
Automatically generated - may contain errors
Lab products found in correlation
Geldoc it2, by Analytik Jena (2 mentions)
Rna millennium marker ladder, by Thermo Fisher Scientific (2 mentions)
2 rna loading dye, by Thermo Fisher Scientific (2 mentions)
Megaclear transcription clean up kit, by Thermo Fisher Scientific (2 mentions)
Northernmax denaturing gel buffer, by Thermo Fisher Scientific (2 mentions)
Amersham hybond n blot, by GE Healthcare (2 mentions)
Ultrahyb hybridization buffer, by Thermo Fisher Scientific (2 mentions)
Nucleotrap mrna mini kit, by Macherey-Nagel (1 mentions)
4 protocols using northernmax running buffer
1
Assessing RNA Quality via Gel Electrophoresis
2
Assessing RNA Quality via Gel Electrophoresis
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After IVT, RNA was purified using MEGAclear™ Transcription Clean-up Kit (ThermoFisher, UK) according to the manufacturer's protocol. In order to assess the quality of the RNA, purified RNAs and the RNA Millennium Marker Ladder (ThermoFisher, UK) were mixed with 2Â RNA loading dye (ThermoFisher, UK) and incubated at 50 °C for 30 min to denature the RNA. A 1.2% agarose gel with 1Â NorthernMax Running Buffer (ThermoFisher, UK) was prepared. After incubation, the denatured ladder and samples were added to the gel and the gel was run at 80 V for 45 min. The gel was then imaged on a GelDoc-It2 (UVP, UK).
3
RNA Northern Blot Analysis
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Poly A+ RNA was fractionated from total RNA by NucleoTrap mRNA Mini Kit (Macherey-Nagel). 5 μg of Poly A+ RNA from HCT116 WT or KO cells were separated on 1% agarose gel prepared with NorthernMax Denaturing Gel Buffer (Ambion) and run in NorthernMax Running Buffer (Ambion). RNAs were then transferred to Amersham Hybond-N+ blot (GE Healthcare) by capillary transfer in 10 x SSC and crosslinked to the blot by UV (254 nm, 120mJ/cm2).
The DNA probes were labeled with [α−32P] dCTP by Prime-It II Random Primer Labeling Kit (Stratagene) as per manufacturer’s instructions. Hybridization was carried out using ULTRAhyb Hybridization Buffer (Ambion) containing 1 × 106 cpm/ml of denatured radiolabeled probes overnight at 42°C. Blots were then washed with 2 x SSC, 0.1% SDS and 0.1 x SSC, 0.1% SDS sequentially at 42°C, and developed using phosphor-imager.
The DNA probes were labeled with [α−32P] dCTP by Prime-It II Random Primer Labeling Kit (Stratagene) as per manufacturer’s instructions. Hybridization was carried out using ULTRAhyb Hybridization Buffer (Ambion) containing 1 × 106 cpm/ml of denatured radiolabeled probes overnight at 42°C. Blots were then washed with 2 x SSC, 0.1% SDS and 0.1 x SSC, 0.1% SDS sequentially at 42°C, and developed using phosphor-imager.
4
Pre-rRNA Northern Blot Analysis
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For the pre-rRNA Northern, 2 μg of total RNA extracted from WI-38 cells treated with Ctr-ASO or ASO-SNUL was separated on 1% denature agarose gel prepared with NorthernMax Denaturing Gel Buffer (Ambion) and run in NorthernMax Running Buffer (Ambion). RNA was then transferred to Amersham Hybond-N+blot (GE Healthcare) by capillary transfer in 10 x SSC and crosslinked to the blot by UV (254 nm, 120mJ/cm2). The DNA probes were labeled with [α–32P] dCTP by Prime-It II Random Primer Labeling Kit (Stratagene) as per manufacturer’s instructions. Hybridization was carried out using ULTRAhyb Hybridization Buffer (Ambion) containing 1X106 cpm/ml of denatured radiolabeled probes overnight at 42°C . Blots were then washed and developed using phosphor-imager.
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