| Description |
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The 0.5-10 Kb RNA Ladder is suitable for sizing single-stranded RNA fragments from 0.5-10 kb. The ladder is synthesized using in vitro transcription of templates from human and rat genes.
The important features of the ladder are listed below:
- Consists of 9 RNA fragments ranging in the size from 0.5-10 kb
- 1.5 kb reference band is ~2-fold brighter for easy orientation and band size determination
- Designed for use with formaldehyde or glyoxal agarose gels
- Suitable for use as a control template for cDNA synthesis as all RNA fragments in the ladder contain a 40 nucleotide poly A tail
- Visualized with ethidium bromide (EtBr) or SYBRR Green II staining
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| Specifications |
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Storage Buffer: 10 mM HEPES, pH 7.2; 2 mM EDTA |
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| Directions |
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Thaw the 0.5-10 Kb RNA Ladder on ice and mix gently to ensure the ladder is homogenous. Avoid vortexing the ladder.
To avoid staining/destaining steps, you may add RNase-free EtBr solution to a final concentration of 10-50 μg/ml to the ladder.
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Formaldehyde Denaturing Gels |
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- Pre-run a horizontal 1.2-1.5% agarose gel (2-3 mm, lane width) containing 6% (w/v) formaldehyde, 20 mM MOPS (pH 7.0), 1 mM EDTA submerged in buffer (20 mM MOPS, pH 7.0; 1 mM EDTA) for 10 minutes at 100 V.
- To 3 μg (3 μl) of ladder, add loading buffer (recommended final loading buffer concentration is 20 mM MOPS; pH 7.0, 6% w/v formaldehyde, 31% v/v deionized formamide, 1 mM EDTA, 0.0125% xylene cyanol and 0.0125% bromophenol blue) and mix.
- Denature the ladder in loading buffer at 70°C for 10 minutes. Keep on ice for 1-2 minutes to quench.
- Load ladder and your RNA samples on the pre-run gel from Step 1.
- Perform electrophoresis at 100 V until the bromophenol blue dye has migrated two-thirds the gel length.
- Stain the RNA ladder with:
- 0.5 μg/ml EtBr solution for 10 minutes and detain the gel in deionized water for 10 minutes OR
- 1:10000 dilution of SYBR Green II RNA Gel Stain in TBE for 20-40 minutes. No destaining is required.
- Visualize ladder bands on a UV transilluminator (302 nm) and photograph using a gel documentation system.
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The RNA bands may fade and disappear on prolonged exposure to UV |
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