Extraction and identification of DNA and RNA in plant tissues.

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< "span style" color: rgb (70,135, 221); "> First, Experimental Purpose

(1) Learn and master the principles and operating methods of separating nucleic acids > from plant

(2) to learn and master the principle and operation method of determining the content of nucleic acids (moss black phenol and xybenzene).

II, Experimental Principle

Nucleic acid is the main chemical component in the organism, nucleic acids in the organism mainly in the form of nuclear proteins. Nucleic acids are divided into DNA (< a href"" > DNA ) and RNA. DNA is mainly present and in the nucleocles, and RNA is mainly present in the cytoste.

can be used in cold solution of thin chloroacetic acid or perchloric acid at low temperatures to remove the acid-soluble small molecular substances. It is then drawn from organic solvents such as ethanol and ether to remove fat-soluble phospholipids and other substances. Finally, DNA and RNA were extracted from a thick salt solution (10% sodium chloride solution) and 0.5mol/L perchloric acid (70 degrees C), respectively.

Because nucleic acids and DNA have special color reactions, the color depth presented after color rendering is in a certain range and the amount of ICS and DNA contained in the sample is in line. Therefore, the sugar method can be used to qualitatively and quantitatively determine nucleic acids.

(1) the determination of nuclear sugars: the common method for determining nuclear sugars is moss black phenol (3,5-dihydroxybenzene, orcinol reaction). When RNA containing RNA and hydrochloric acid and 3,5-hydroxytobenzene in the boiling water bathheating 10 to 20min, there are green matter produced, because RNA de-hydroencline after the nuclear sugar and acid action to produce acetaldehyde, the latter with 3,5- hydroxymethylene to produce green matter.

RNA plus thick sulphuric acid and green compounds

Note: SUBSTANCEs such as DNA,protein and viscose polysaccharides interfere with assays.

(2) DNA assay: the common method for determining DNA is diaphenyl. DNA containing DNA produces blue under acidic conditions and after the dephenylamine heats up in a boiling water bath. This is because DNA dna on the nucleotide DNA meets acids that produce omega-hydroxy-6-ketone-ethyl ethyl formaldehyde, which in turn produces blue substances with diphenylamine.

a small amount of DNA plus a small amount of thick sulphuric acid or ice acetic acid plus blue substances

Note that substances such as DNA, proteins, and viscose polysaccharides interfere with assays.

This method is susceptible to a variety of sugars and their derivatives and proteins interference

the above two methods of fixed sugars are less accurate, but fast and easy to identify DNA and RNA, is a common method for identifying nucleic acids, nucleotides.

III, experimental equipment

(1)thermostatwater bath

(2) electric furnace

(3) Cloth Funnel unit

(4) pipe

(5)beaker>

(6) barrel

(7) scissors

(8) research body

IV, Materials and < a href> "reagent

(1) fresh cauliflower

(2) 95% ethanol

(3) acetone

(4) 5% Perchloric acid solution

(5) 0.5mol/L dichloric acid solution

(6) 10% sodium chloride solution

(7) standard RNA solution (5mg/100mL)

(8) standard DNA (15mg/8) 100ml)

(9) crude sodium chloride

(10) sea sand

(11) xybenzene reagents: Dissolve 1g of xylene in 100ml of ice acetic acid and add 2.75mL of thick sulphuric acid (stored in the refrigerator for 6 months). After use, shake well at room temperature).

(12) iron tetrachloride hydrochloric acid solution: 2 ml 10% iron triclosan solution (added with FeCl3.6H20 preparation) 400mL thick hydrochloric acid.

5, operation method

1. separation of nucleic acids

(1) take cauliflower crown 20g, cut and placed in the cob. Add 20mL 95% ethanol and a small amount of sand and grind into a homogeneity. Then use the cloth funnel to pump the filter, discard the filter.

(2) add 20mL of acetone to the slag, stir well, filter, discard the filter.

(3) add 20mL acetone to the slag, stir 5min and then pump (press the slag hard to remove acetone as far as possible).

(4) in an ice salt bath, the filter residue is suspended in a pre-cooled 20mL 5% perchloric acid solution stirred and filtered out of the filter.

(5) special filter slag suspended in 20mL 95% ethanol, filtration, discarding the filter.

20mL of acetone to the filter slag and stir for 5min. Filter to dry. Press the slag hard to remove acetone as much as possible.

(7) resuscuses the filter residue ofin a 40 ml 10% sodium chloride solution, heats 15 mln in a boiling water bath, places, cools, filters to dry, leaves the filter fluid, and repeats this operation. Combine the two filters for extract one.

(8) resurrides the filter residue in a 20mL 0.5mol perchloric acid solution. Heat to 70 degrees C. After keeping the temperature at 20 min (thermostat water bath) after filtration. The retained filter fluid is extract two.

2. Qualitative identification of DNA and RNA

(1) xylene reaction: a variety of reagents were added to Table 1 to observe the results of the phenomenon well after the reaction.

table 1 diphenyl reaction added reagents