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Description: The
GenElute Mammalian Total RNA Purification Kit combines silica-membrane technology
with a convenient spin column format for a rapid bind, wash, and elute method
to prepare high quality total RNA
(Figure 1).
Samples are lysed and homogenized in guanidine thiocyanate and 2-mercaptoethanol
to release RNA and inactivate RNases. Lysates are spun through a filtration
column to remove cellular debris and shear DNA. The filtrate is then
applied to a high capacity silica column to bind total RNA, followed by washing
and elution. Up to 150 µg of total RNA can be recovered per prep in 100 µl
of water. The purified RNA is ready for Northern blot analysis
(Figure 1), RT-PCR (Figure 2) and other common applications.
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Protocol for GenElute Mammalian
Total RNA Purification Kit |
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Features and Benefits
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Versatile
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Purify total RNA from up to 107 cells or 40 mg of tissue.
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High yields
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Up to 150 µg of pure, concentrated total RNA per prep
in
50 - 100 µl of water.
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Efficient
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Recover RNA from as few as 100 cells.
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Fast
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12 to 18 preps in 30 minutes without lengthy gravity flow or
ultracentrifugation steps.
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Safe
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No toxic solvents such as phenol/chloroform.
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Cost effective
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40% more purifications than the leading supplier.
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GenElute Mammalian Total RNA Purification Kits
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Product Code
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Name
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Purifications per kit
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Technical Manual .pdf
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Short protocols .pdf
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GenElute Mammalian Total RNA Purification |
10
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GenElute Mammalian Total RNA Purification
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70
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GenElute Mammalian Total RNA Purification
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350
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Kit Contents
- Lysis Solution
- b-mercaptoethanol
- Wash Solution 1
- Wash Solution 2
- Elution Solution
- Filtration Columns with Tubes
- RNA Binding Spin Columns with Tubes
- Collection Tubes
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High quality RNA from various tissues and cells.
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Figure 1. Formaldehyde-agarose gel and Northern blot of total RNA purified with the
GenElute Mammalian Total RNA Purification Kit.
Upper panel: 2 µg of each RNA analyzed on a 1.2% agarose gel containing
0.6M formaldehyde. Lower panel: Corresponding Northern blot hybridized
with a 32P labeled RNA probe for GAPDH in PerfectHybTM Plus hybridization buffer. Note: The GAPDH probe detected a single mRNA band in every lane with little or no smearing. Even RNA from pancreas, which is known to have high RNase levels, is not degraded.
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RNA purification using Sigma's GenElute Mammalian Total RNA Isolation Kit is suitable for RT-PCR.
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Figure 2.
RT-PCR analysis of RNA isolated with the GenElute Mammalian Total RNA
Purification Kit. RT-PCR detection with RNA from 100 to 1,000 cells. HeLa cells were diluted to
give 0, 100, 300, or 1,000 cells per tube followed by RNA purification using the
GenElute Mammalian Total RNA Isolation Kit. Duplicate 10 µl samples (20%)
of each preparation were treated with Amplification Grade DNase I. One of each pair was reverse transcribed with
Enhanced Avian Reverse Transcriptase (+ lanes). The other was incubated
under the same conditions, but without the reverse transcriptase (- lanes). PCR
was performed with 2 µl (10%) from each reaction, GAPDH primers, and Taq DNA Polymerase. One-fifth of each PCR product was fractionated
on a 1.5% agarose gel, and photographed after staining for 30 minutes with SybrGreen I. RT-PCR products are clearly visible for all reactions with
reverse transcriptase added (+ lanes), except those with no cells (Lane 0). No PCR
products are visible for reactions without reverse transcriptase added (- lanes),
demonstrating that RT-PCR products with reverse transcriptase are from RNA and
not from residual, contaminating DNA. |
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