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Acridine orange staining for ssDNA

Acridine orange staining for ssDNA - Basic Lab Protocols and Techniques

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Old 10-12-2010, 09:22 AM
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Default Acridine orange staining for ssDNA



Hallo All,
I separated ssDNA in denatured PAGE gels. I want to extract the bands for micro arrays. Silver staining is not a good method when extracting bands. Please suggest a protocol for Acridine orange staining of ssDNA in denatured PAGE gels.
I silver stained the gels, but i dont know, if i can extract the bands and use them for micro arrays.
Please help me in this regard,
Greetings,
Harish.
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Old 02-25-2011, 04:15 PM
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Default Re: Acridine orange staining for ssDNA

Acridine orange can be used when is necessary to differentiate between single-stranded and double-stranded DNA in a gel, that under short-wave UV irradiation, double-stranded DNA fluoresces is green, while single-stranded DNA giving a red fluoresces. When increased ratios of dye to DNA are used, all molecules fluoresce green. This stain is not ideal for everyday use because the background fluorescence can often be high, leading to low signal-to-noise ratio and decreased sensitivity of detection.
Reagent
Acridine orange stock solution : dissolve 10 mg/ml acridine orange in distilled water, and store in glass amber bottle, protected from light at 4 C.
Procedure
After electrophoretic run, remove gel from apparatus and place in adapted enamel container. Cover to a depth of 5 mm with diluted acridine orange solution at 30 g/ml (for 100 ml stain solution, dilute 0.3 ml acridine orange stock solution up to 100 ml with distilled water). Stain the gel in the dark for 30 min., then destain, again in the dark, for 30 min. in distilled water. Removal of high background fluorescence can be achieved by destaining for 16 hrs at 4 C in the dark. If destaining is incomplete, the background will appear very green and the red bands will appear dull or even black. Faint bands may not be visible at this stage. View under 300 nm UV radiation, and photograph gel is best carried out with a red photographic filter. The red filter enhances the discrimination of the red bands against the green background.
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Old 02-25-2011, 06:40 PM
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Default Re: Acridine orange staining for ssDNA

Quote:
Originally Posted by harish View Post
I silver stained the gels, but i dont know, if i can extract the bands and use them for micro arrays.
I believe you can. Silver ions have positive charge so they stick to the negative charged part of DNA: the phosphate groups. Considering this, you should have no problem with microarrays since silver is not messing (theorically) with the bases, ergo do not mess with binding of target DNA with the probes.
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