※ Principle : mini spin column (silica matrix)
※ Sample size : up to 100 mg plant tissue or 1 x107 plant cells
※ Operation time : 30 ~ 60 minutes
※ Binding capacity : up to 100 μg total RNA / column
※ Expected yield : 5 ~ 30 μg of total RNA from 100 mg of young leave
※ Column applicability : centrifugation and vaccum
※ Minimum elution volume : 30 μl
Important Notes :
※ Make sure everything is RNase-free when handling RNA.
※ Buffers provided in this system contain irritants. Wear gloves and lab coat when handling these buffers.
※ Caution: ß-mercaptoethanol is hazardous to human health. Perform the procedures involving RB Buffer or PRB Buffer in a chemical fume hood.
※ Add required volume of RNase-free ethanol (96~100%) to Wash Buffer 2 when first use.
※ Dilute RNase-free DNase 1 in reaction buffer (1M NaCl, 10 mM MnCl2, 20 mM Tris-HCl, pH 7.0 at 25ºC) to final conc. = 0.5 U/μl.
Problems |
Possible reasons |
Solutions |
Low or no yield of RNA |
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Incorrect preparation of Wash Buffer 2. |
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Wash Buffer 2 is not mixed with ethanol before use. |
Make sure that the correct volumes of ethanol (96- 100 %) is added into Wash Buffer 2 when first opened. |
The volume or the percentage of ethanol is not correct before adding into Wash Buffer 2. |
Make sure that the correct volumes of ethanol (96- 100 %) is added into Wash Buffer 2 when first used. |
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Elution of genomic RNA is not efficient. |
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RNase-free water is not completely absorbed by column membranes. |
After RNase-free water is added, stand the RB Column for 1 min before centrifugation. |
Column is clogged |
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Sample is too viscous. |
Reduce the sample volume. |
Degradation of eluted RNA |
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Sample is old. |
Always use fresh samples. |