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An extraction method and chloroplast technology, which are applied in the field of plant genetic engineering, can solve the problems of low purity of chloroplast organelles, reduced yield of chloroplast DNA, and low yield of chloroplast organelles, and achieve simple and fast extraction methods, saving operation time and high yield high effect
Inactive Publication Date: 2012-10-17
ZHEJIANG MARINE DEV RES INST
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Because algal cell walls are relatively thick and not easy to break, the yield of chloroplast organelles is low, and the tissue is rich in polysaccharides and polyphenols, the purity of isolated chloroplast organelles is not high
The report also involves the step of dialysis of chloroplast DNA solution to remove CsCl, which can easily lead to a decrease in the yield of chloroplast DNA and prolong the extraction time
Using the currently reported algae chloroplast extraction methods, the extraction rate of chloroplast DNA is generally 10-15 μg / g (fresh algae), and the extraction rate is low
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Embodiment 1
[0032] 1) Raw material preparation: select fresh wakame algae, wash and drain, weigh 20g of algae, add 150ml of buffer A, and use a tissue homogenizer at 0-4°C to evenly grind the algae liquid;
[0033] 2) The homogenized algae liquid was filtered with 60-mesh nylon cloth, and the filtrate was centrifuged at 4,000×g for 10 minutes to obtain the precipitate;
[0034] 3) Add 15ml of buffer B to the collected centrifuged precipitate at 65°C for 1 hour in a water bath;
[0035] 4) The lysate was treated with phenol-chloroform-isoamyl alcohol (volume ratio 25:24:1) and chloroform-isoamyl alcohol (volume ratio 24:1) to remove denatured proteins, and CsCl (final concentration 1.45g / ml) and Hoechst 33258 (final concentration 10μg / ml), centrifuge at 380,000×g for 8h;
[0036] 5) Use a syringe to quickly remove the band containing chloroplast DNA under ultraviolet light irradiation, and remove Hoechst 33258 with isopropanol saturated with 1M NaCl;
[0037] 6) Add 3M sodium acetate, do...
Embodiment 2
[0047] 1) Raw material preparation: select fresh Ulva algae bodies, wash and drain, weigh 25g of algae bodies, add 150ml of buffer A, and use a tissue homogenizer at 0-4°C to evenly grind them into algae liquid;
[0048] 2) The homogenized algae liquid was filtered with 60-mesh nylon cloth, and the filtrate was centrifuged at 4,000×g for 10 minutes to obtain the precipitate;
[0049] 3) Add 20ml of buffer B to the collected centrifuged precipitate, at 65°C, in a water bath for 1.5h;
[0050] 4) The lysate was treated with phenol-chloroform-isoamyl alcohol (volume ratio 25:24:1) and chloroform-isoamyl alcohol (volume ratio 24:1) to remove denatured proteins, and CsCl (final concentration 1.35g / ml) and Hoechst 33258 (final concentration 10μg / ml), centrifuge at 380,000×g for 8h;
[0051] 5) Use a syringe to quickly remove the band containing chloroplast DNA under ultraviolet light irradiation, and remove Hoechst 33258 with isopropanol saturated with 1M NaCl;
[0052] 6) Add 3M ...
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Abstract
The invention relates to a method for extracting a DNA of an algae chloroplast. The method comprises the following steps of carrying out pulp homogenization of fresh or frozen algae as a raw material by a tissue pulp homogenization machine, adding a buffer solution A into the homogenate obtained by the previous step, carrying out centrifugation by a centrifugal force of 3000 to 6000xg, collecting precipitates, adding a buffer solution B into the precipitates, carrying out water bath pyrolysis at a temperature of 60 to 70 DEG C for 1 to 2 hours, removing denatured proteins of the lysate, adding CsCl and Hoechst 33258 into the lysate, carrying out centrifugation by a centrifugal force of 36000 to 40000xg for 8 to 12 hours, taking out a stripe containing a DNA of an algae chloroplast, removing Hoechst 33258, adding 2.5 to 3.5M of sodium acetate, double distilled water and isopropanol into the stripe, standing at a temperature of -20 DEG C for 20 to 40 minutes, carrying out centrifugation by a centrifugal force of 12000 to 16000xg for 15 to 30 minutes to obtain precipitates, and carrying out purification to obtain the DNA of the algae chloroplast. Compared with the prior art, the method simplifies extraction processes, has simple and feasible processes, improves an extraction ratio and purity of a DNA of an algae chloroplast, realizes an algae (fresh algae) chloroplast DNA yield of 150 to 200 micrograms per gram, and can fully satisfy requirements of conventional molecular biology processes such as genomic sequencing, specific enzyme digestion, random amplified polymorphic DNA (RAPD), polymerase chain reaction (PCR) and target gene sequence cloning.
Description
technical field [0001] The invention relates to the technical field of plant genetic engineering and specifically designs a method for extracting macroalgae chloroplast DNA with high efficiency and high purity. Background technique [0002] Chloroplast is an extremely important organelle for photosynthesis in plants, and it has independently replicated genetic material. Chloroplast DNA (cpDNA) is widely used in studies of cytoplasmic inheritance, plant male sterility, plant phylogeny, genetic diversity and kinship. Due to the important functions of chloroplasts and their DNA, they have always been important research objects in cell biology, genetics and molecular biology. Due to the difficulty in obtaining a sufficient amount of intact chloroplasts to obtain high-purity chloroplast DNA (cpDNA), the progress of this research has been slow. At present, many methods are obtained by crushing plants such as homogenization, ultrasonic crushing, freeze-thawing and chemical method...
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