sample into each well after mixing the aliquot with fast orange tracking dye. Run a ladder in one lane. The gel should be electrophoresed at 40-50 volts, stained in ethidium bromide, and visualized under an ultraviolet light source. For even clearer gels, use the formaldehyde electrophoresis protocol.
More Info:
-
This protocol adapted from Wilkins, T. A. and L. B. Smart (1996). Isolation of RNA from plant tissues in Krieg, P. A. (ed.) A Laboratory Guide to RNA: Isolation, Analysis, and Synthesis. Wiley-Liss, Inc.: 21-40.
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This procedure has produced decent yields of RNA from cotton tissues, a genus that does not easily yield nucleic acids due to polysaccharides, phenolics, etc.
-
Yield is approximately 100-500 µg/g tissue.
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Yield results are best using cotyledons, mid-range for leaves and whole ovules, and low for fibers. Typical results are given in the table below.
|
Sample Tissue |
Mass |
|
|
|
|
A2 leaves |
0.3 g |
1.898 |
97 |
322 |
|
A2 cotyledons |
0.3 g |
1.910 |
125 |
417 |
|
D5 leaves |
0.3 g |
1.800 |
48 |
160 |
|
D5 ovules, 10 dpa |
0.3 g |
1.750 |
89 |
222 |
|
D5 ovules, 20dpa |
0.4 g |
1.813 |
33 |
82.5 |
|
AD3 ovules |
0.23g |
1.968 |
115 |
500 |
Reagents and Solutions
DEPC-Treated Water
< !> Perform all these steps under a fume hood!
Add 0.05% (v/v) DEPC to the required volume of distilled, deionized water (for example, 500 mL of water requires the addition of 250µL DEPC). Mix by vigorous stirring for approximately 30 minutes. Autoclave to break down the DEPC.
Hot Borate Extraction Buffer (XT Buffer)
Absolute For 100 mL
0.2M Borax (Sodium-borate decahydrate) 7.63 grams
30 mM EGTA 1.14 grams
1% (w/v) SDS 1.0 gram
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