CN105112401A - Method for efficiently extracting midge larva DNA (deoxyribonucleic acid) - Google Patents
Method for efficiently extracting midge larva DNA (deoxyribonucleic acid) Download PDFInfo
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- CN105112401A CN105112401A CN201510563452.5A CN201510563452A CN105112401A CN 105112401 A CN105112401 A CN 105112401A CN 201510563452 A CN201510563452 A CN 201510563452A CN 105112401 A CN105112401 A CN 105112401A
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Abstract
The invention discloses a method for efficiently extracting midge larva DNA (deoxyribonucleic acid), which comprises the following steps: taking a single midge larva, pulling out and discarding all intestinal tracts with a dissecting needle to the greatest extent, immersing the larva in distilled water, putting the obtained larva in a 1.5ml Ep tube, adding a lysis solution, and sufficiently grinding the larva with a grinding rod; adding 20 mu l of proteinase K and 15 mu l of ribonuclease, uniformly mixing, putting in a 56-DEG C water bath, and carrying out digestive treatment over night; extracting with chloroform and isoamyl alcohol twice; adding 300 mu l of saturated phenol and 300 mu l of chloroform; extracting with isoamyl alcohol once; finally, adding 0.5 volume of isopropanol ice, and precipitating; and washing with 70% ethanol, drying at 37 DEG C in a drying oven, and adding 30 mu ldd of H2O (37 DEG C) to dissolve the DNA. The method can obtain the midge larva high-quality DNA, and the midge larva high-quality DNA can be directly used in PCR (polymerase chain reaction) to amplify the COI gene.
Description
The present invention obtains Tianjin Natural Science Fund In The Light (14JCQNJC14600), state natural sciences fund (31101653,31272284), Tianjin S & T Developmentin High Institutions fund (20090608) and the funded projects such as Tianjin Normal University's technician introduction fund (5RL104), the outstanding young teacher's funded project (2013) of Students' Innovation foundation drill program (201410065046) and Tianjin.
Technical field
The invention belongs to Animal molecular biology technical field, particularly a kind of method for high efficiency extraction chironomus larvas DNA.
Background technology
Midge is under the jurisdiction of Insecta Diptera Nemocera (Diptera:Nematocera), and the adult bodily form is similar to mosquito, has the title of " the midge worm not stinging people " because its mouthpart is degenerated.Because of adult static time front foot do not stop dynamic and gain the name, therefore be called midge.Midge and human lives closely bound up.Ecological demand and the life habit of Notes On The Chironomidae Larvae there are differences because of the difference of kind, different to environmental factor susceptibility, are one of excellent indicator organisms of monitoring water environment.In addition, in aquatic products is produced, midge as the bait of economic fish, can have very high economic worth.
Midge is that complete metamorphosis grows insect, and multiple species larva and the problem such as pupa plesiomorphism, same species different developmental phases data deficiency are all that the species identification of midge brings puzzlement.Since DNA bar code technology proposed from 2003, development rapidly, is widely used in vegeto-animal taxonomic identification.The acquisition of bar code data generally comprises following step: extract STb gene, pcr amplification goal gene, and agarose gel electrophoresis and trace of albumin nucleic acid instrument detect, and purifying is reclaimed in rubber tapping, order-checking, these steps for the treatment of and analysis data.Wherein, extract this step of STb gene very crucial, concentration and the purity of extracting STb gene directly have influence on subsequent experimental.Traditional method (as CTAB, NaCl, SDS method) and RNA isolation kit can be adopted when extracting DNA.Traditional method and RNA isolation kit cut both ways, but in extraction chironomus larvas DNA, the two effect is all not good.
Summary of the invention
The object of this invention is to provide a kind of method of high efficiency extraction chironomus larvas DNA.For reaching this target, what the present invention adopted is increase pre-treatment, and is improved on the basis of original traditional method, thus can obtain high-quality chironomus larvas DNA.The technical solution adopted in the present invention is:
A method of high efficiency extraction chironomus larvas DNA, is characterized in that being undertaken by following step:
(1) pre-treatment is increased: get single chironomus larvas, all pulled out by its enteron aisle as far as possible with dissecting needle and discard; Use distilled water immersion polypide, every 5min changes first water, changes twice and amounts to 15min;
(2) placed in 1.5mlEp pipe by (1) gained larva, add lysate, grind with grinding rod to larva, grinding fully;
(3) add 20ul Proteinase K and 15ulRNA enzyme, mixing, is placed in 56 DEG C of water-baths and carries out digested overnight process;
(4) chloroform of 24:1 by volume: isoamyl alcohol extraction twice;
(5) the saturated phenol of 300 μ l, 300 μ l chloroforms are added: isoamyl alcohol extraction once;
(6) add 0.5 volume isopropanol to precipitate on ice;
(7) 70% washing with alcohol twice;
(8) 37 DEG C, baking oven oven dry, adds 30ulddH
2o(37 DEG C) DNA is dissolved.Wherein lysate (100ml) is composed as follows:
CTAB2g final concentration 2%
SDS0.5g final concentration 0.5%
0.5MEDTA6ml final concentration 0.03M
1MTris-HCl10ml final concentration 0.1M
5MNaCl20ml final concentration 1M
PVP1g final concentration 1%
Beta-mercaptoethanol 0.1ml final concentration 0.1%
Distilled water is mended to 100ml.
The method of high efficiency extraction chironomus larvas DNA of the present invention, can obtain high quality DNA in single chironomus larvas body, can be directly used in PCR reaction and amplify Mitochondrial cytochrome c oxidase subunit I gene (COI gene).
The present invention is more detailed to be described below:
(1) pre-treatment is increased: get single chironomus larvas, when doing head capsule load, all pulled out by its enteron aisle as far as possible with dissecting needle and discard, prevent the digested thing of enteron aisle from causing interference to subsequent experimental; Use distilled water immersion polypide, every 5min changes first water, changes twice and amounts to 15min, to remove the impurity on polypide.
(2) placed in 1.5mlEp pipe by 1 gained larva, add lysate, grind with grinding rod to larva, grinding fully.
(3) add 20ul Proteinase K and 15ulRNA enzyme, mixing, is placed in 56 DEG C of water-baths and carries out digested overnight process.Just starting in the 2h of heating in water bath, every 10min manually puts upside down mixing to sample and shakes up once.
(4) add 450ul chloroform: primary isoamyl alcohol (24:1), fully mixes, leave standstill the centrifugal 20min of 10min, 12000rpm, get supernatant.Repeat this operation once.
(5) add the saturated phenol of 300 μ l, 300 μ l chloroforms: primary isoamyl alcohol, fully mixing of turning upside down, leaves standstill 10min, centrifuging and taking supernatant.
(6) add 0.5 volume isopropanol, mixing, precipitate 30min on ice, the centrifugal 20min of 12000rpm, abandons supernatant.
(7) add 600ul70% ethanol, the centrifugal 20min of 12000rpm, abandons supernatant.Repeat once.
(8) 37 DEG C, baking oven oven dry, adds 30ulddH
2o(37 DEG C) DNA is dissolved.
(9) pcr amplification COI gene go forward side by side row agarose gel electrophoresis detect.
The high spot reviews of the present invention configuration of lysate and the digestion time of Proteinase K and RNA enzyme are on the impact of extracting chironomus larvas DNA, chironomus larvas protein content height increases Protein Extraction number of times to the impact of extracting chironomus larvas DNA, therefore solves the problem of chironomus larvas DNA extraction poor quality.
The positively effect that chironomus larvas DNA extraction method disclosed by the invention is compared with prior art had is:
(1) cost is low.Lysate composition all has commercially available, and oneself deployment cost ratio test kit cost is low.
(2) quality is good.Extract chironomus larvas DNA by this method, pcr amplification COI gene success ratio is high.
Accompanying drawing explanation
Fig. 1 pcr amplification COI gene.
Embodiment
Below in conjunction with embodiment, the present invention is described, the scheme of embodiment described here, do not limit the present invention, one of skill in the art can make improvements and change according to spirit of the present invention, these described improvement and change all should be considered as within the scope of the invention, and scope of the present invention and essence are limited by claim.Various raw material used all has commercially available:
Embodiment 1: chironomus larvas DNA extraction
Preparation lysate (100ml):
CTAB2g final concentration 2%
SDS0.5g final concentration 0.5%
0.5MEDTA6ml final concentration 0.03M
1MTris-HCl10ml final concentration 0.1M
5MNaCl20ml final concentration 1M
PVP1g final concentration 1%
Beta-mercaptoethanol 0.1ml final concentration 0.1%
Distilled water is mended to 100ml.
(1) pre-treatment:
A. get single chironomus larvas, when doing head capsule load, as far as possible its enteron aisle is all pulled out with dissecting needle and discard, prevent the digested thing of enteron aisle from causing interference to subsequent experimental.
B. use distilled water immersion polypide, every 5min changes first water, changes twice and amounts to 15min, to remove the impurity on polypide.Finally with thieving paper, polypide surface distilled water is blotted.
(2) placed in 1.5mlEp pipe by 1 gained larva, add 200ul lysate, grind with sterilized grinding rod to larva, grinding fully.Add 200ul lysate again and rinse grinding rod.
(3) add 20ul Proteinase K and 15ulRNA enzyme, mixing, is placed in 56 DEG C of water-baths and carries out digested overnight process.Just starting in the 2h of heating in water bath, every 10min manually puts upside down mixing to sample and shakes up once.
(4) add 450ul chloroform: primary isoamyl alcohol (24:1), fully mixes, leave standstill the centrifugal 20min of 10min, 12000rpm, get supernatant.
(5) (4) are repeated.
(6) add the saturated phenol of 300 μ l, 300 μ l chloroforms: primary isoamyl alcohol, fully mixing of turning upside down, leaves standstill 10min, centrifuging and taking supernatant.
(7) add 0.5 volume isopropanol, mixing, precipitate 30min on ice, the centrifugal 20min of 12000rpm, abandons supernatant.
(8) add 600ul70% ethanol, the centrifugal 20min of 12000rpm, abandons supernatant.Repeat once.
(9) 37 DEG C, baking oven oven dry, adds 30ulddH
2o(37 DEG C) DNA is dissolved.The DNA product obtained is placed in the preservation of-20 DEG C, refrigerator.
Embodiment 2:
Pcr amplification Mitochondrial cytochrome c oxidase subunit I gene (COI gene)
(1) PCR reaction system:
Total system 25ul:ddH
2o16.2ul, 10 X PCRBuffer2.5ul, dNTPs (2.5mmol/L) 2ul, universal primer LCO and HCO (10umol/L) each 1ul, Taq enzyme (500U, 2.5U/ul) 0.3ul, DNA profiling 2ul.
(2) PCR reaction conditions:
94 DEG C of denaturation 5min; 94 DEG C of sex change 40s; 56 DEG C of annealing 40s; 72 DEG C extend 1min; 30 circulations; 72 DEG C extend 5min; 4 DEG C of preservations.
(3) configure 1% sepharose and carry out electrophoresis detection, see accompanying drawing.
Result illustrates:
(1) electrophorogram the 1st swimming lane is 8KpMarker, the 2nd, 3,4,5 swimming lanes are respectively single and only redly nakedly must extract PCR result after DNA by chironomus larvas.
(2) COI gene is about 698bp, and between Marker the 1st, 2 bands, result meets expection.
Embodiment 3
Simultaneous test
Conclusion: comparatively ordinary method DNA concentration is high for method of the present invention, and purity is high.
Claims (3)
1. a method of high efficiency extraction chironomus larvas DNA, is characterized in that being undertaken by following step:
(1) pre-treatment is increased: get single chironomus larvas, all pulled out by its enteron aisle as far as possible with dissecting needle and discard, prevent the digested thing of enteron aisle from causing interference to subsequent experimental; Use distilled water immersion polypide, every 5min changes first water, changes twice and amounts to 15min, to remove the impurity on polypide;
(2) placed in 1.5mlEp pipe by (1) gained larva, add lysate, grind with grinding rod to larva, grinding fully;
(3) add 20ul Proteinase K and 15ulRNA enzyme, mixing, is placed in 56 DEG C of water-baths and carries out digested overnight process;
(4) chloroform of 24:1 by volume: isoamyl alcohol extraction twice;
(5) the saturated phenol of 300 μ l, 300 μ l chloroforms are added: isoamyl alcohol extraction once;
(6) add 0.5 volume isopropanol to precipitate on ice;
(7) 70% washing with alcohol twice;
(8) 37 DEG C, baking oven oven dry, adds 30ulddH
2o(37 DEG C) DNA is dissolved.
2. the method for high efficiency extraction chironomus larvas DNA described in claim 1, wherein lysate (100ml) is composed as follows:
CTAB2g final concentration 2%
SDS0.5g final concentration 0.5%
0.5MEDTA6ml final concentration 0.03M
1MTris-HCl10ml final concentration 0.1M
5MNaCl20ml final concentration 1M
PVP1g final concentration 1%
Beta-mercaptoethanol 0.1ml final concentration 0.1%
Distilled water is mended to 100ml.
3. described in claim 1, the method for high efficiency extraction chironomus larvas DNA is characterized in that: can obtain high quality DNA in single chironomus larvas body, can be directly used in PCR reaction and amplify Mitochondrial cytochrome c oxidase subunit I gene (COI gene).
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107881167A (en) * | 2017-12-14 | 2018-04-06 | 重庆师范大学 | A kind of mosquito foot sample DNA extracting method and system |
| CN109735631A (en) * | 2019-01-22 | 2019-05-10 | 湖北民族学院 | A kind of pair of stick arteries and veins midge gene 16SrDNA fragment sequence combines the analysis method of its form |
| CN111575277A (en) * | 2020-06-04 | 2020-08-25 | 天津师范大学 | Method for extracting molting DNA (deoxyribonucleic acid) in pupal stage of single chironomidae |
| CN114540256A (en) * | 2022-04-14 | 2022-05-27 | 天津师范大学 | Method for separating and culturing intestinal bacteria of chironomidae insects |
| CN114574481A (en) * | 2022-04-06 | 2022-06-03 | 天津师范大学 | Method for effectively extracting chironomid larva transcriptome gene |
| CN114574480A (en) * | 2022-04-06 | 2022-06-03 | 天津师范大学 | Method for extracting molting total DNA of water surface mixed chironomidae insects |
Citations (1)
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| CN102994493A (en) * | 2012-11-06 | 2013-03-27 | 中国科学院南海海洋研究所 | Method for extracting total DNA (deoxyribonucleic acid) from body wall of sea cucumber |
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2015
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102994493A (en) * | 2012-11-06 | 2013-03-27 | 中国科学院南海海洋研究所 | Method for extracting total DNA (deoxyribonucleic acid) from body wall of sea cucumber |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107881167A (en) * | 2017-12-14 | 2018-04-06 | 重庆师范大学 | A kind of mosquito foot sample DNA extracting method and system |
| CN109735631A (en) * | 2019-01-22 | 2019-05-10 | 湖北民族学院 | A kind of pair of stick arteries and veins midge gene 16SrDNA fragment sequence combines the analysis method of its form |
| CN111575277A (en) * | 2020-06-04 | 2020-08-25 | 天津师范大学 | Method for extracting molting DNA (deoxyribonucleic acid) in pupal stage of single chironomidae |
| CN111575277B (en) * | 2020-06-04 | 2023-10-24 | 天津师范大学 | Method for extracting single molting DNA in midge period of midge family |
| CN114574481A (en) * | 2022-04-06 | 2022-06-03 | 天津师范大学 | Method for effectively extracting chironomid larva transcriptome gene |
| CN114574480A (en) * | 2022-04-06 | 2022-06-03 | 天津师范大学 | Method for extracting molting total DNA of water surface mixed chironomidae insects |
| CN114574480B (en) * | 2022-04-06 | 2024-03-01 | 天津师范大学 | Method for extracting total DNA of mixed insect molting of midge on surface of water body |
| CN114540256A (en) * | 2022-04-14 | 2022-05-27 | 天津师范大学 | Method for separating and culturing intestinal bacteria of chironomidae insects |
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Application publication date: 20151202 |