GB/T 19564-2004 Experimental method for identification of larch seeds - Random amplified polymorphic DNA method
Some standard content:
ICS 65.020.20
National Standard of the People's Republic of China
GB/T 19564--2004
Experimental identification method for species of larch seed
Random amplified polymorphic DNA method
Experimental identification method for species of larch seedRAPD2004-06-22Promulgated
General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of ChinaStandardization Administration of China
2004-12-01Implementation
Appendix A of this standard is a normative appendix.
This standard is proposed by the General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China. GB/T19564—2004
Drafting units of this standard: National Agricultural Standardization Monitoring and Research Center (Heilongjiang), Heilongjiang Provincial Quality and Technical Supervision Bureau. Main drafters of this standard: Wang Qinggui, Xu Jing, Jiang Wen, Li Guangyu, Shi Shaoye, Zhu Weiguo. Before the introduction of GB/T19564-2004, the identification of forest seeds in my country was mostly done by sensory identification, physical and chemical identification and other methods. These methods require a long inspection cycle and do not have many advantages of being stable. With the development of molecular biology, especially since the 1990s, molecular biology related technologies have been widely used, and the detection object is TNA macromolecules, the genetic basis of organisms. Using LNA as the detection object has many advantages that other detection methods do not have: first, the number of DNA markers available for detection may be unlimited, which is incomparable to interferon technology; second, DNA analysis technology does not vary with tissues or developmental stages like other technologies. DNA provided from any part of the plant body and at any time can be used for analysis, and the detection results are the same; third, LNA analysis is not affected by the environment. Its variation only comes from the variation of the allele DNA sequence. This stability makes it easy to reveal the genetic variation between varieties, thereby excluding the phenotypic variation caused by environmental variation. Based on the above advantages, DVA analysis technology is an advanced method for variety identification in agriculture, forestry, animal husbandry, fishery, etc. 1 Scope
Experimental method for identification of larch seeds
Random amplified polymorphic DNA method
This standard specifies the specific experimental method for identification of larch seeds. GB/T19564—2004
This standard applies to the experimental process of identification of larch seeds using the random amplified polymorphic DNA (RAPD) method. 2 Terms, abbreviations and abbreviations
2.1 Terms and definitions
The following terms and definitions apply to this standard. 2.1.1
Polymerase chain reaction-an enzyme-catalyzed reaction that can amplify a DNA fragment of a specific base sequence from as few as 10 copies to millions of molecules in vitro in a few hours, that is, a DNA synthesis reaction. 2. 1.2
Randomly amplified polymorphic DNA is a molecular marker therapy based on polymerase chain reaction, using randomly arranged oligodeoxynucleotide single strands as primers to amplify IDNA at different sites in the genome, thereby revealing polymorphisms between alleles at multiple sites. 2. 1.3
Nucleotide
is the basic unit of nucleic acid, composed of three parts: pentose, phosphate and cyclic base. 2. 1. 4
Primer
A short single strand complementary to the template DNA chain, providing the 3'-OH end as the starting point of DNA synthesis, extending the complementary chain of the synthetic template DNA.
Polymorphism
A pair of primers amplifies between two or more different genomic DNA materials to obtain DNA fragments of different numbers or lengths.
2.2 Abbreviations
The following abbreviations apply to this standard.
RAPD Random Amplified Polymorphism DNA PCR Polymerase Chain Reaction DNA Deoxyribonucleic Acid OD Optical Density
RNA Ribonucleic Acid TBE Tris Boric-acid EDTA KAO Ni KAca
GB/T19564—2004
3 Experimental Methods
3.1 Principles
DNA is the hereditary substance of organisms, carrying all the reverse hereditary information. Identification at the DNA molecular level is the most effective method to distinguish the differences between species, varieties and even individuals. PCR technology is used to amplify DNA fragments by several times or even millions of times in a short period of time, so as to achieve the purpose of detection. The RAPD method uses random nucleotide sequences as primers based on PCR technology to amplify genomic DNA. The polymorphism of these amplified DNA fragments reflects the polymorphism of DNA in the corresponding region of the genome. According to the differences in the length and number of amplified fragments, a certain species and variety can be identified.
3.2 Environmental conditions
a) Temperature. 15℃～25℃,
b) Relative humidity: Relative humidity (RH) is not more than 50%. 3.3 Instruments
a) PCR amplification instrument;
b) UV spectrophotometer:
High-speed desktop centrifuge: speed not less than 150001/minld) Multi-purpose electrophoresis instrument and horizontal electrophoresis tank; e)
UV radiator
Micropipette: specifications are 0.1μL~2.5μL, 2μL-20μL, 20xL200μL, 100μL1000μLf
Electronic balance: graduation value is 0.000 1#g)
Magnetic heating stirrer:
Gel imaging system;
Ultrapure water system;
k) Sterilizer,
Acidity meter: the maximum allowable error is ±0.1pHm)
Microwave oven
n) Constant temperature water bath: the maximum allowable error is ±1C; o) Constant temperature drying oven, the maximum allowable error is ±1℃. 3.4 Reagents and consumables
3.4.1 Reagents
) TagDNA polymerase: storage condition is -20℃ ± 2℃; b) 10×Buffer: storage condition is -20=2℃; four deoxynucleotides (4×dNTP): storage condition is -20℃ ± 2℃; d) Mg2+: storage condition is 20℃ ± 2℃; RNase (no DNase): storage condition is →20℃ ± 2℃; e) Agarose:
g) Tris: molecular formula is C4HnNO, disodium ethylenediaminetetraacetate (EDTANa·2HzO): molecular formula is CeHuN,OaNaz·2H,Oh)
Bromphenol blue sodium salt: molecular formula is CiH,Br, O, SNa ;) Xylene cyanole FF: molecular formula is CsHNzOS, Nak) 8-Hydroxyquinoline (hydroxyyuinoline): molecular formula is CH, NO: 1)
Sodium dodecyl sulfate (SDS): molecular formula is CHasO, SNa; m)
Ethidium disulfate (EB): molecular formula is C; HN, Brn) Isoquinoline: molecular formula is C, HlOH;
Crystallization (pheno1). Molecular formula is CH. O, storage conditions are -20℃±2℃; p) Chloroform: molecular formula is CHCls, purity is analytical grade g)
Boric acid: molecular formula is H,BOs, purity is analytical grade; r) Sucrose: molecular formula is C:H22Oi, purity is analytical grade; Anhydrous ethanol: molecular formula is CH,OH, purity is analytical grade, s)
Hydrochloric acid: molecular formula is HCI, purity is analytical grade Sodium fluoride: molecular formula is NaCI, purity is analytical grade; u)
v) Water: molecular formula is H,0. All water used in this experiment is deionized water. 3. 4.2 Consumables
GB/T 19564—2004
a) Centrifuge tube: specification is 1.5 mL, 0.2 rnL, high pressure sterilization is required before use (operate according to the method specified in Chapter A.1):
Grinding body: specifications are 7 cm~10 cm in diameter
c) Pipette tip: specifications are 20μL, 200μI, and 1(00μL. High pressure sterilization is required before use: d)
Volume flask: specifications are 100mL, 1000mL; beaker: specifications are 100ml-:
) Erlenmeyer flask: specifications are 250ml;
g) PE gloves,
h) Radiant paper;
) Sealing film.
3.5 Experimental Procedure
3.5.1 DNA Extraction
3.5.1.1 Take 5~8 larch seeds, place them in a 1.5ml centrifuge tube, add 600μL homogenization buffer (prepared according to the method specified in Chapter A.2), soak for 2h, pour into a mortar and grind thoroughly, pour the ground product into a 1.5mL centrifuge tube, take a small amount of homogenization buffer to rinse the mortar, pour into the centrifuge tube, centrifuge (speed 4000 r/min) for 10 min, transfer the supernatant to a new 1.5mL centrifuge tube, and discard the precipitate.
3.5.1.2 Add saturated phenol (prepared according to the method specified in Chapter A.3) with the same volume as the supernatant, and slowly invert the centrifuge tube for 20 min to avoid vigorous shaking.
3.5.1.3 Centrifuge at a speed of 8000 r/min for 10 min and transfer the supernatant to a new centrifuge tube. 3.5.1.4 Add an equal volume of phenol-trifluoromethane-isoamyl alcohol (volume ratio of 24:23=1) to the supernatant, centrifuge at a speed of 8 000 r/min for 10 min, and transfer the supernatant to a new centrifuge tube. 3.5.1.5 Add an equal volume of trifluoromethane-isoamyl alcohol (volume ratio of 23:1) to the supernatant, slowly invert for 10 min, centrifuge at a speed of 8 00 r/min for 10 min, and transfer the supernatant to a new centrifuge tube. 3.5.1.6 Add twice the volume of ice-cold ethanol as the supernatant, rotate the centrifuge tube horizontally for 50-100 times, and white flocculent DNA will appear. 3.5.1.7 Place the centrifuge tube in a 20℃ refrigerator for 30 minutes, then centrifuge (speed 8000r/min) for 10 minutes to form a white precipitate. 3.5.1.8 Weigh the iridium in the centrifuge tube, add 1 mL 75% ethanol, centrifuge (speed 15 000 z/min) for 5 minutes, pour out the ethanol, pour it on clean water-resistant paper, and absorb the liquid. 3.5.1.9 Place the centrifuge tube in a constant temperature drying oven (40℃50℃) for 20 minutes or in a ventilated place for 40 minutes to evaporate the ethanol. 3.5.1.10 Add 100)uI.TF buffer (prepared according to the method specified in Chapter A.4) and 2μL of RNase (prepared according to the method specified in Chapter A.5), water bath (55±2℃) for 12 hours to fully dissolve the DNA precipitate, and store in ice at 4C. 3.5.2 DNA concentration measurement
3.5.2.1 Absorption test
The oxazine ring and pyrimidine ring in DNA molecules can absorb ultraviolet light. The ultraviolet absorption peak of DNA is at a wavelength of 260 nm, and the absorption peak of protein is at a wavelength of 280 nm. Take 5μL of the DNA solution extracted in 3.5.1, add 0.95μL of water, put it in a colorimetric cup, and use a TiKAMiKAca
GB/T19564-2004
ultraviolet spectrophotometer to detect, record the OD values at 260 nm and 280 nm, calculate the DNA concentration and the ratio of OD2u and OD28. The OD2u/OD of DNA is preferably around 1.8, and above 1.5 can also be used for RAPD-PCR analysis. If the ratio is too small, repeat steps 3.5.1.2 to 3.5.1.10 to continue extraction. DNA concentration is calculated according to formula (1);
D=50×100×ODauXs
Where;
D--DNA concentration, in nanograms per microliter (ng/μL); s--dilution factor.
3.5.2.2 Gel electrophoresis detection
Put the prepared liposaccharide gel (prepared according to the method specified in Chapter A.6) with a concentration of 7 μg/μL into the electrophoresis insert, so that the sample well is at the negative end of the power supply, and add enough electrophoresis buffer (0.5×TBE) (prepared according to the method specified in A.6.3) to just cover the gel surface to a depth of 1 mmol. Take 10 μL of DNA solution and 2 μL of gel sample buffer (prepared according to the method specified in Chapter A.7), pipette up and down on the sealing film to mix, and add to the sample well (avoid bubbles). Turn on the power supply, control the voltage at 5V/cm, electrophoresis for 1h, take out the gel, and place it on a UV transilluminator for viewing. If the DNA is of good quality, molecular weight (>50 μb) and not degraded, a uniform dense band will appear near the spotting hole. If the DNA is partially degraded, it will be distributed continuously. If it is severely degraded, no large DNA fragments can be seen, and RNA can only be detected far away from the spotting hole. 3.5.3 RAFD amplification
3.5.3.1 Reaction components
25μL reaction system contains 1×Buffer, 1,5ttnal/LMg+, 200μnol/L dNTP, 1UTa DNA polymerase, 1μmol/L primer, 100 nM template DNA, add water to 25L (the preparation of each reaction component is carried out according to the method specified in Chapter A, 8). Add the reaction solution into a 0.2mL centrifuge tube in sequence according to the above proportions, put it into the PCR instrument, and perform PCR cycles. 3.5.3.2 Cycling process
The cycling process is shown in Figure 1:
Pre-denaturation at 94°C for 5 min
Denaturation at 94°C for 1 minwwW.bzxz.Net
Incubation at 36°C for 1 min
Extension at 72°C for 1 min
Extension at 72°C for 10 tnin
Cooling at 4°C
40 cycles
Figure 1 Schematic diagram of the cycling process of PCR reaction
3.5.3.3 Electrophoresis detection
Put the prepared 2% agarose gel (prepared according to the method specified in Section A.6) into the electrophoresis medium so that the sample spot is at the negative end of the power supply. Take 10L of PCR amplification product and 2L of gel-avoiding sample flushing solution (prepared according to the method specified in Chapter A, 7), pipette up and down on the sealing film to mix, and add to the spotting hole (avoid bubbles). Turn on the power supply and control the voltage at 3 V/cr, electrophoresing for 3 hours, take out the gel, and scan it on the gel imaging. 3.5.3.4 Monitoring and identification
Compare the gel imaging results of the variety to be tested with the standard spectrum bands of the seeds of each distribution area of the variety, so as to identify the authenticity of the variety.
A.1 Operation method of high pressure sterilization
Appendix A
(Normative Appendix)
Sterilization method and preparation of buffer and main reagents CB/T19564—2004
Put the centrifuge arm to be sterilized into a beaker and seal it with tin foil paper; put the pipette tip into the tip box: put the prepared reagent into the reagent bottle, cover it tightly, and put it into the sterilizer. First, increase the pressure to 1Pa, release the air, and then increase the pressure to 1Pa again. Start timing. After 20 minutes, turn off the power. When the pressure is zero, take out the centrifuge, tip and reagent bottle for standby use. A.2 Preparation of homogenization buffer
Take 200mL of 1mol/1.Tris·CI (pH8.0), 50mL of 0.5mol/LEDTA (pH8.0) and 0.5gSDS, and add water to 100L. Before use, ensure that SDS is fully dissolved. If crystals appear, place it in a 50℃ water bath to dissolve for 10 minutes. A.2.1 1 mol/L Tris-Cl (pH8.0): weigh 12.11 g Tris, dissolve in 80 mL water, adjust the pH value of the solution to 8.0 with concentrated HCl, add water to make up to 100 tml. Autoclave. A.2.2 0.5 mol/L EDTA (pH8.0): weigh 18.61 g EDTANaz2H2O, dissolve in 80 mL water, stir vigorously with a magnetic heating stirrer, adjust the pH value of the solution to 8.0 with NaOH (about 2 g), add water to make up to 100 mL, and autoclave. A.3 Preparation of saturated
The preparation of saturated phenol should be carried out in a well-ventilated or well-ventilated environment. A.3.1Put the phenol at room temperature in a 60℃ water bath to melt, then pour it into a distiller to heat, and collect the liquid at about 180℃. The redistilled phenol is divided and stored in a refrigerator at 20℃ for later use. A3.2After redistilled phenol is melted in a 60℃ water bath, add 8-hydroxyquinoline to reach a final concentration of 0.1%, add an equal volume of 0.5moi/LT'ris-CI (pH8.0) solution, mix with a magnetic heating stirrer, let it stand for a while, remove the upper aqueous phase, and repeat this process until the pH value of the phase is greater than 7.8, which is a saturated phenol solution, put it into a brown bottle and store it in a refrigerator at 4℃. A.4 Preparation of TE buffer
Take 1 mL of 1 mol/L Tris-Cl (pH7.6), 0.5 mol/L FDTA (pH8.0), 0.2 mL, add water to 100 mLs4.4.11 mol/L Tris·Cl (pH7.6): weigh 12.11 Tris+ and dissolve in 80 mL of water, adjust the pH value of the solution to 7.6 with concentrated HC, add water to 100 mL, and sterilize by high pressure. A.4.2 0.5 mol/L EDTA (pH8.0): A.2.2A.5 Preparation of RNase (10 mg/mL)
Weigh 1 mg of RNase and dissolve in 100 μL of TE buffer, incubate in water (70°C) for 10 min and slowly cool to room temperature. A.6 Preparation of agarose gel
To prepare agarose gel with a concentration of 0.7% (or 2%), weigh 0.7g (or 2g) of agarose and place it in a 200ml conical flask, dissolve it in 100ml 0.5×TBF, and dissolve it in a microwave oven until the solution is transparent. Cool it to 50℃-60℃, add ethidium bromide (10mg/mI.) and adjust it to a final concentration of 0.5ug/mL. After mixing, pour the gel onto a sealed and combed gel plate (about 1.0II from the bottom plate). The thickness of the gel should be 3mm-5mm. After the gel is completely solidified, put it into the electrophoresis tank for use. A6.1 Ethyl bromide (EB stock solution (10 mg/ml), weigh 1g of EB, dissolve it in 1.00ml of water, stir it with a magnetic boron thermo stirrer for several hours to ensure that it is completely dissolved, transfer it to a brown bottle, and store it in a refrigerator at 4℃. KAO Ni KAca
GB/T 19564--2004
A, 6.25×TBE weigh 54g of Tris in 800mL of water, add 20mL of 0.5mol/LEDTA (pH8.0) and 27.5g of acid, dilute to 1000mL, sterilize by high pressure, and store at room temperature. A.6.30.5×TBE weigh 100mL of 5×TBE, dilute to 1000mL with water, and set aside. A, 7 Gel loading buffer
0.25% phenol blue, 0.25% dimethoate FF, 40% (mass concentration) sucrose aqueous solution. A8 Preparation of reaction components in the reaction system A 8. 11 U TqDNA incubation buffer
The volume of TaqDNA synthase to be added in the 25L reaction system is calculated according to formula (A.1): Voo
Wherein, V—volume of TaqDNA synthase, in microliters (μl). D—concentration of TaqDNA synthase, in units per microliter (U/μL). A.8.21.5mmol/LMg2+
The volume of Mg*+ added in the 25uL reaction system is calculated according to formula (A2): 1, 5 X 25
Where:
VM+ volume, unit is microliter<(μL);
D—Mg\+ concentration, unit is millimole per liter (mmol/L). A, 8. 310 μmol/1, Primer
First centrifuge the primer, then dilute with water. Add the volume calculated according to formula (A, 3), store in a 20℃ refrigerator, dilute three times with water before each use, and the final concentration is 10 μmol/L. OD2 × 33
Where M in formula (A, 3) is calculated according to formula (A.4) 1M = nA X 313.22+nG× 329. 22+nCX 289.19+nT × 304. 19 -61. 97 Where:
water volume, unit is microliter (L);
M—primer molecular weight, unit is gram per liter (/mL); ni—number of bases;
A—adenine;
guanine:
C. cytosine;
T—thymine.
Art References
GB/T19564—2004
[1] J. Samblick, EF Fritsch, T. Maniatis. Molecular cloning, Beijing, Science Press, 1996[2] Benjamin Levwin. Genes. New Yark: Oxford University Press, 2000[3J Jain, A. , C. Apparanda, PL, Bhalla. Evaluation of genetic diversity and genome finger-printing of Pandorea (Bignoniaceae) by RAPD and inter-SSR PCR. Genome 42, 1999. 714~71[4 Kujimn. T. , T. Nagaoka, K. Noda, et al. Genetic linkage map nf ISSR and RAPD markers in Einkorn wheat in relation to that of RFLP markers, Theor. Appl. Genct. 96, 1998.37~45[5] Lanham. PG, R, M. Brennan. Genetic characterizatian af gooseherry (Ribes grossulariasubgenus Grossularia) germplasm using RAPD, ISSR and AFLP markers. J Hort Sci Biotech 74,1999.361~366
[6] MeGregor. CE,C. A, Lambert, MM Greyling, et al. A cotnparative assessment ofDNA fingerprinting techniques (RAPD, ISSR, AF1.P and SSR) in tetraploid potato (Solanum tuberosutn L,)germplasm. Euphytica 113, 2000. 135~144[?] Naganka, T., Y. Ogihara. Applicability of inter-simple sequence repeat polymorphisms inwheat for use as DNA markers in comparison to RFL.P and RAPD markers. Theor Appi Genet 94,1997.597~602
E8J Willinas JGK et al. DNA polymorphism anplificarion by arbitrary primers are useful as genetic markers.Nucleic AcidsRes,,1990.18(4):6531-6535KANiKAca0) solution, mix with a magnetic heating stirrer and let it stand for a while, remove the upper aqueous phase, repeat this process until the pH value of the phase is greater than 7.8, which is a saturated phenol solution, put it into a brown bottle and store it in a refrigerator at 4℃. A. Preparation of 4TE buffer
Take 1 mol/L Tris-Cl (pH7.6) 1 mL, 0.5 mol/I FDTA (pHII8.0) 0.2 mL, add water to 100 mLs4.4.11mol/1Tris·Cl (pH7.6): weigh 12.11Tris+ and dissolve it in 80mL water, adjust the pH value of the solution to 7.6 with concentrated HC, add water to 100 tniL, and sterilize by high pressure. A 4.2 0.5 mol/L EDTA (pH8.0): A. 2.2A. 5 Preparation of RNase (10 mg/mL)
Weigh 1 mg of RNase and dissolve it in 100 μL TE buffer. Incubate at 70°C for 10 min and then slowly cool to room temperature. A.6 Preparation of agarose gel
To prepare agarose gel with a concentration of 0.7% (or 2%), weigh 0.7g (or 2g) of agarose and place it in a 200ml conical flask, dissolve it in 100ml 0.5×TBF, and dissolve it in a microwave oven until the solution is transparent. Cool it to 50℃-60℃, add ethidium bromide (10mg/mI.) and adjust it to a final concentration of 0.5ug/mL. After mixing, pour the gel onto a sealed and combed gel plate (about 1.0II from the bottom plate). The thickness of the gel should be 3mm-5mm. After the gel is completely solidified, put it into the electrophoresis tank for use. A6.1 Ethyl bromide (EB stock solution (10 mg/ml), weigh 1g of EB, dissolve it in 1.00ml of water, stir it with a magnetic boron thermo stirrer for several hours to ensure that it is completely dissolved, transfer it to a brown bottle, and store it in a refrigerator at 4℃. KAO Ni KAca
GB/T 19564--2004
A, 6.25×TBE weigh 54g of Tris in 800mL of water, add 20mL of 0.5mol/LEDTA (pH8.0) and 27.5g of acid, dilute to 1000mL, sterilize by high pressure, and store at room temperature. A.6.30.5×TBE weigh 100mL of 5×TBE, dilute to 1000mL with water, and set aside. A, 7 Gel loading buffer
0.25% phenol blue, 0.25% dimethoate FF, 40% (mass concentration) sucrose aqueous solution. A8 Preparation of reaction components in the reaction system A 8. 11 U TqDNA incubation buffer
The volume of TaqDNA synthase to be added in the 25L reaction system is calculated according to formula (A.1): Voo
Wherein, V—volume of TaqDNA synthase, in microliters (μl). D—concentration of TaqDNA synthase, in units per microliter (U/μL). A.8.21.5mmol/LMg2+
The volume of Mg*+ added in the 25uL reaction system is calculated according to formula (A2): 1, 5 X 25
Where:
VM+ volume, unit is microliter<(μL);
D—Mg\+ concentration, unit is millimole per liter (mmol/L). A, 8. 310 μmol/1, Primer
First centrifuge the primer, then dilute with water. Add the volume calculated according to formula (A, 3), store in a 20℃ refrigerator, dilute three times with water before each use, and the final concentration is 10 μmol/L. OD2 × 33
Where M in formula (A, 3) is calculated according to formula (A.4) 1M = nA X 313.22+nG× 329. 22+nCX 289.19+nT × 304. 19 -61. 97 Where:
water volume, unit is microliter (L);
M—primer molecular weight, unit is gram per liter (/mL); ni—number of bases;
A—adenine;
guanine:
C. cytosine;
T—thymine.
Art References
GB/T19564—2004
[1] J. Samblick, EF Fritsch, T. Maniatis. Molecular cloning, Beijing, Science Press, 1996[2] Benjamin Levwin. Genes. New Yark: Oxford University Press, 2000[3J Jain, A. , C. Apparanda, PL, Bhalla. Evaluation of genetic diversity and genome finger-printing of Pandorea (Bignoniaceae) by RAPD and inter-SSR PCR. Genome 42, 1999. 714~71[4 Kujimn. T. , T. Nagaoka, K. Noda, et al. Genetic linkage map nf ISSR and RAPD markers in Einkorn wheat in relation to that of RFLP markers, Theor. Appl. Genct. 96, 1998.37~45[5] Lanham. PG, R, M. Brennan. Genetic characterizatian af gooseherry (Ribes grossulariasubgenus Grossularia) germplasm using RAPD, ISSR and AFLP markers. J Hort Sci Biotech 74,1999.361~366
[6] MeGregor. CE,C. A, Lambert, MM Greyling, et al. A cotnparative assessment ofDNA fingerprinting techniques (RAPD, ISSR, AF1.P and SSR) in tetraploid potato (Solanum tuberosutn L,)germplasm. Euphytica 113, 2000. 135~144[?] Naganka, T., Y. Ogihara. Applicability of inter-simple sequence repeat polymorphisms inwheat for use as DNA markers in comparison to RFL.P and RAPD markers. Theor Appi Genet 94,1997.597~602
E8J Willinas JGK et al. DNA polymorphism anplificarion by arbitrary primers are useful as genetic markers.Nucleic AcidsRes,,1990.18(4):6531-6535KANiKAca0) solution, mix with a magnetic heating stirrer and let it stand for a while, remove the upper aqueous phase, repeat this process until the pH value of the phase is greater than 7.8, which is a saturated phenol solution, put it into a brown bottle and store it in a refrigerator at 4℃. A. Preparation of 4TE buffer
Take 1 mol/L Tris-Cl (pH7.6) 1 mL, 0.5 mol/I FDTA (pHII8.0) 0.2 mL, add water to 100 mLs4.4.11mol/1Tris·Cl (pH7.6): weigh 12.11Tris+ and dissolve it in 80mL water, adjust the pH value of the solution to 7.6 with concentrated HC, add water to 100 tniL, and sterilize by high pressure. A 4.2 0.5 mol/L EDTA (pH8.0): A. 2.2A. 5 Preparation of RNase (10 mg/mL)
Weigh 1 mg of RNase and dissolve it in 100 μL TE buffer. Incubate at 70°C for 10 min and then slowly cool to room temperature. A.6 Preparation of agarose gel
To prepare agarose gel with a concentration of 0.7% (or 2%), weigh 0.7g (or 2g) of agarose and place it in a 200ml conical flask, dissolve it in 100ml 0.5×TBF, and dissolve it in a microwave oven until the solution is transparent. Cool it to 50℃-60℃, add ethidium bromide (10mg/mI.) and adjust it to a final concentration of 0.5ug/mL. After mixing, pour the gel onto a sealed and combed gel plate (about 1.0II from the bottom plate). The thickness of the gel should be 3mm-5mm. After the gel is completely solidified, put it into the electrophoresis tank for use. A6.1 Ethyl bromide (EB stock solution (10 mg/ml), weigh 1g of EB, dissolve it in 1.00ml of water, stir it with a magnetic boron thermo stirrer for several hours to ensure that it is completely dissolved, transfer it to a brown bottle, and store it in a refrigerator at 4℃. KAO Ni KAca
GB/T 19564--2004
A, 6.25×TBE weigh 54g of Tris in 800mL of water, add 20mL of 0.5mol/LEDTA (pH8.0) and 27.5g of acid, dilute to 1000mL, sterilize by high pressure, and store at room temperature. A.6.30.5×TBE weigh 100mL of 5×TBE, dilute to 1000mL with water, and set aside. A, 7 Gel loading buffer
0.25% phenol blue, 0.25% dimethoate FF, 40% (mass concentration) sucrose aqueous solution. A8 Preparation of reaction components in the reaction system A 8. 11 U TqDNA incubation buffer
The volume of TaqDNA synthase to be added in the 25L reaction system is calculated according to formula (A.1): Voo
Wherein, V—volume of TaqDNA synthase, in microliters (μl). D—concentration of TaqDNA synthase, in units per microliter (U/μL). A.8.21.5mmol/LMg2+
The volume of Mg*+ added in the 25uL reaction system is calculated according to formula (A2): 1, 5 X 25
Where:
VM+ volume, unit is microliter<(μL);
D—Mg\+ concentration, unit is millimole per liter (mmol/L). A, 8. 310 μmol/1, Primer
First centrifuge the primer, then dilute with water. Add the volume calculated according to formula (A, 3), store in a 20℃ refrigerator, dilute three times with water before each use, and the final concentration is 10 μmol/L. OD2 × 33
Where M in formula (A, 3) is calculated according to formula (A.4) 1M = nA X 313.22+nG× 329. 22+nCX 289.19+nT × 304. 19 -61. 97 Where:
water volume, unit is microliter (L);
M—primer molecular weight, unit is gram per liter (/mL); ni—number of bases;
A—adenine;
guanine:
C. cytosine;
T—thymine.
Art References
GB/T19564—2004
[1] J. Samblick, EF Fritsch, T. Maniatis. Molecular cloning, Beijing, Science Press, 1996[2] Benjamin Levwin. Genes. New Yark: Oxford University Press, 2000[3J Jain, A. , C. Apparanda, PL, Bhalla. Evaluation of genetic diversity and genome finger-printing of Pandorea (Bignoniaceae) by RAPD and inter-SSR PCR. Genome 42, 1999. 714~71[4 Kujimn. T. , T. Nagaoka, K. Noda, et al. Genetic linkage map nf ISSR and RAPD markers in Einkorn wheat in relation to that of RFLP markers, Theor. Appl. Genct. 96, 1998.37~45[5] Lanham. PG, R, M. Brennan. Genetic characterizatian af gooseherry (Ribes grossulariasubgenus Grossularia) germplasm using RAPD, ISSR and AFLP markers. J Hort Sci Biotech 74, 1999.361~366
[6] MeGregor. CE, C. A, Lambert, MM Greyling, et al. A cotnparative assessment ofDNA fingerprinting techniques (RAPD, ISSR, AF1.P and SSR) in tetraploid potato (Solanum tuberosutn L,)germplasm. Euphytica 113, 2000. 135~144[?] Naganka, T., Y. Ogihara. Applicability of inter-simple sequence repeat polymorphisms inwheat for use as DNA markers in comparison to RFL.P and RAPD markers. Theor Appi Genet 94,1997.597~602
E8J Willinas JGK et al. DNA polymorphism anplificarion by arbitrary primers are useful as genetic markers.Nucleic AcidsRes,,1990.18(4):6531-6535KANiKAca2A. 5 Preparation of RNA enzyme (10 mg/mL)
Weigh 1 mg of RNA enzyme and dissolve it in 100 μl TE buffer, incubate at 70°C for 10 min and slowly cool it to room temperature. A. 6 Preparation of agarose gel
To prepare agarose gel with a concentration of 0.7% (or 2%), weigh 0.7 g (or 2 g) of agarose and place it in a 200 ml conical flask, dissolve it in 100 ml 0.5×TBF, and dissolve it in a microwave oven until the solution is transparent. Cool it to 50°C-60°C, add ethidium bromide (10 mg/ml) and adjust it to a final concentration of 0.5 ug/mL. After mixing, pour the gel onto a sealed and combed plate (about 1.0 μl from the bottom plate). The gel thickness should be 3 mm to 5 mm. After the gel is completely solidified, put it into the electrophoresis tank for use. A6.1 Ethyl bromide (EB stock solution (10 mg/ml), weigh 1g of EB, dissolve it in 1.00ml of water, stir it with a magnetic boron thermo stirrer for several hours to ensure that it is completely dissolved, transfer it to a brown bottle, and store it in a refrigerator at 4℃. KAO Ni KAca
GB/T 19564--2004
A, 6.25×TBE weigh 54g of Tris in 800mL of water, add 20mL of 0.5mol/LEDTA (pH8.0) and 27.5g of acid, dilute to 1000mL, sterilize by high pressure, and store at room temperature. A.6.30.5×TBE weigh 100mL of 5×TBE, dilute to 1000mL with water, and set aside. A, 7 Gel loading buffer
0.25% phenol blue, 0.25% dimethoate FF, 40% (mass concentration) sucrose aqueous solution. A8 Preparation of reaction components in the reaction system A 8. 11 U TqDNA incubation buffer
The volume of TaqDNA synthase to be added in the 25L reaction system is calculated according to formula (A.1): Voo
Wherein, V—volume of TaqDNA synthase, in microliters (μl). D—concentration of TaqDNA synthase, in units per microliter (U/μL). A.8.21.5mmol/LMg2+
The volume of Mg*+ added in the 25uL reaction system is calculated according to formula (A2): 1, 5 X 25
Where:
VM+ volume, unit is microliter<(μL);
D—Mg\+ concentration, unit is millimole per liter (mmol/L). A, 8. 310 μmol/1, Primer
First centrifuge the primer, then dilute with water. Add the volume calculated according to formula (A, 3), store in a 20℃ refrigerator, dilute three times with water before each use, and the final concentration is 10 μmol/L. OD2 × 33
Where M in formula (A, 3) is calculated according to formula (A.4) 1M = nA X 313.22+nG× 329. 22+nCX 289.19+nT × 304. 19 -61. 97 Where:
water volume, unit is microliter (L);
M—primer molecular weight, unit is gram per liter (/mL); ni—number of bases;
A—adenine;
guanine:
C. cytosine;
T—thymine.
Art References
GB/T19564—2004
[1] J. Samblick, EF Fritsch, T. Maniatis. Molecular cloning, Beijing, Science Press, 1996[2] Benjamin Levwin. Genes. New Yark: Oxford University Press, 2000[3J Jain, A. , C. Apparanda, PL, Bhalla. Evaluation of genetic diversity and genome finger-printing of Pandorea (Bignoniaceae) by RAPD and inter-SSR PCR. Genome 42, 1999. 714~71[4 Kujimn. T. , T. Nagaoka, K. Noda, et al. Genetic linkage map nf ISSR and RAPD markers in Einkorn wheat in relation to that of RFLP markers, Theor. Appl. Genct. 96, 1998.37~45[5] Lanham. PG, R, M. Brennan. Genetic characterizatian af gooseherry (Ribes grossulariasubgenus Grossularia) germplasm using RAPD, ISSR and AFLP markers. J Hort Sci Biotech 74,1999.361~366
[6] MeGregor. CE,C. A, Lambert, MM Greyling, et al. A cotnparative assessment ofDNA fingerprinting techniques (RAPD, ISSR, AF1.P and SSR) in tetraploid potato (Solanum tuberosutn L,)germplasm. Euphytica 113, 2000. 135~144[?] Naganka, T., Y. Ogihara. Applicability of inter-simple sequence repeat polymorphisms inwheat for use as DNA markers in comparison to RFL.P and RAPD markers. Theor Appi Genet 94,1997.597~602
E8J Willinas JGK et al. DNA polymorphism anplificarion by arbitrary primers are useful as genetic markers.Nucleic AcidsRes,,1990.18(4):6531-6535KANiKAca2A. 5 Preparation of RNA enzyme (10 mg/mL)
Weigh 1 mg of RNA enzyme and dissolve it in 100 μl TE buffer, incubate at 70°C for 10 min and slowly cool it to room temperature. A. 6 Preparation of agarose gel
To prepare agarose gel with a concentration of 0.7% (or 2%), weigh 0.7 g (or 2 g) of agarose and place it in a 200 ml conical flask, dissolve it in 100 ml 0.5×TBF, and dissolve it in a microwave oven until the solution is transparent. Cool it to 50°C-60°C, add ethidium bromide (10 mg/ml) and adjust it to a final concentration of 0.5 ug/mL. After mixing, pour the gel onto a sealed and combed plate (about 1.0 μl from the bottom plate). The gel thickness should be 3 mm to 5 mm. After the gel is completely solidified, put it into the electrophoresis tank for use. A6.1 Ethyl bromide (EB stock solution (10 mg/ml), weigh 1g of EB, dissolve it in 1.00ml of water, stir it with a magnetic boron thermo stirrer for several hours to ensure that it is completely dissolved, transfer it to a brown bottle, and store it in a refrigerator at 4℃. KAO Ni KAca
GB/T 19564--2004
A, 6.25×TBE weigh 54g of Tris in 800mL of water, add 20mL of 0.5mol/LEDTA (pH8.0) and 27.5g of acid, dilute to 1000mL, sterilize by high pressure, and store at room temperature. A.6.30.5×TBE weigh 100mL of 5×TBE, dilute to 1000mL with water, and set aside. A, 7 Gel loading buffer
0.25% phenol blue, 0.25% dimethoate FF, 40% (mass concentration) sucrose aqueous solution. A8 Preparation of reaction components in the reaction system A 8. 11 U TqDNA incubation buffer
The volume of TaqDNA synthase to be added in the 25L reaction system is calculated according to formula (A.1): Voo
Wherein, V—volume of TaqDNA synthase, in microliters (μl). D—concentration of TaqDNA synthase, in units per microliter (U/μL). A.8.21.5mmol/LMg2+
The volume of Mg*+ added in the 25uL reaction system is calculated according to formula (A2): 1, 5 X 25
Where:
VM+ volume, unit is microliter<(μL);
D—Mg\+ concentration, unit is millimole per liter (mmol/L). A, 8. 310 μmol/1, Primer
First centrifuge the primer, then dilute with water. Add the volume calculated according to formula (A, 3), store in a 20℃ refrigerator, dilute three times with water before each use, and the final concentration is 10 μmol/L. OD2 × 33
Where M in formula (A, 3) is calculated according to formula (A.4) 1M = nA X 313.22+nG× 329. 22+nCX 289.19+nT × 304. 19 -61. 97 Where:
water volume, unit is microliter (L);
M—primer molecular weight, unit is gram per liter (/mL); ni—number of bases;
A—adenine;
guanine:
C. cytosine;
T—thymine.
Art References
GB/T19564—2004
[1] J. Samblick, EF Fritsch, T. Maniatis. Molecular cloning, Beijing, Science Press, 1996[2] Benjamin Levwin. Genes. New Yark: Oxford University Press, 2000[3J Jain, A. , C. Apparanda, PL, Bhalla. Evaluation of genetic diversity and genome finger-printing of Pandorea (Bignoniaceae) by RAPD and inter-SSR PCR. Genome 42, 1999. 714~71[4 Kujimn. T. , T. Nagaoka, K. Noda, et al. Genetic linkage map nf ISSR and RAPD markers in Einkorn wheat in relation to that of RFLP markers, Theor. Appl. Genct. 96, 1998.37~45[5] Lanham. PG, R, M. Brennan. Genetic characterizatian af gooseherry (Ribes grossulariasubgenus Grossularia) germplasm using RAPD, ISSR and AFLP markers. J Hort Sci Biotech 74, 1999.361~366
[6] MeGregor. CE, C. A, Lambert, MM Greyling, et al. A cotnparative assessment ofDNA fingerprinting techniques (RAPD, ISSR, AF1.P and SSR) in tetraploid potato (Solanum tuberosutn L,)germplasm. Euphytica 113, 2000. 135~144[?] Naganka, T., Y. Ogihara. Applicability of inter-simple sequence repeat polymorphisms inwheat for use as DNA markers in comparison to RFL.P and RAPD markers. Theor Appi Genet 94,1997.597~602
E8J Willinas JGK et al. DNA polymorphism anplificarion by arbitrary primers are useful as genetic markers.Nucleic AcidsRes,,1990.18(4):6531-6535KANiKAca7 Gel loading buffer
0.25% phenol blue, 0.25% dimethoate FF, 40% (mass concentration) sucrose aqueous solution. A8 Preparation of reaction components in reaction system A 8. 11 U TqDNA incubation medium
The volume of TagDNA synthase required in 25L reaction system is calculated according to formula (A.1): Voo
Wherein,
V—volume of TaqDNA synthase, in microliter (μL); D—concentration of TaqDNA synthase, in units per microliter (U/μL). A.8.21.5mmol/LMg2+
The volume of Mg+ added in 25uL reaction system is calculated according to formula (A2): 1, 5 X 25
Wherein,
V—volume of TaqDNA synthase, in microliter (μL);
D—concentration of Mg+, in millimole per liter (mmol/L). A, 8. 310 μmol/1, Primer
First centrifuge the primer, then dilute with water. The added volume is calculated according to formula (A, 3), stored in a 20℃ refrigerator, and diluted three times with water before each use, the final concentration is 10 μmol/L. OD2 × 33
Wherein, M in formula (A,3) is calculated according to formula (A.4): 1M = nA X 313.22+nG× 329.22+nCX 289.19+nT × 304.19 -61.97Wherein:
water volume, in microliters (L);
M—primer molecular weight, in grams per liter (/mL); ni—number of bases;
A—adenine;
guanine;
C. cytosine;
T—thymine.
Art Examination Literature
GB/T19564—2004
[1]J. Sambuke, EF Fritsch, T. Maniatis. Molecular cloning, Beijing, Science Press, 1996[2] Benjamin Levwin. Genes .NewYark:Oxford University Press,2000[3J Jain, A. , C. Apparanda, PL, Bhalla. Evaluation of genetic diversity and genome finger-printing of Pandorea (Bignoniaceae) by RAPD and inter-SSR PCR. Genome 42, 1999. 714~71[4 Kujimn. T. , T. Nagaoka, K. Noda, et al. Genetic linkage map nf ISSR and RAPD markersin Einkorn wheat in relation to that of RFLP markers, Theor. Appl. Genct. 96, 1998.37~45[5] Lanham. PG, R, M. Brennan. Genetic characterizatian af gooseherry (Ribes grossulariasubgenus Grossularia) germplasm using RAPD, ISSR and AFLP markers. J Hort Sci Biotech 74, 1999.361~366
[6] MeGregor. CE, C. A, Lambert, MM Greyling, et al. A cotnparative assessment ofDNA fingerprinting techniques (RAPD, ISSR, AF1.P and SSR) in tetraploid potato (Solanum tuberosutn L,)germplasm. Euphytica 113, 2000. 135~144[?] Naganka, T. , Y. Ogihara. Applicability of inter-simple sequence repeat polymorphisms inwheat for use as DNA markers in comparison to RFL.P and RAPD markers. Theor Appi Genet 94,1997.597~602
E8J Willinas JGK et al. DNA polymorphism anplificarion by arbitrary primers are useful as genetic markers.Nucleic AcidsRes,,1990.18(4):6531-6535KANiKAca7 Gel loading buffer
0.25% phenol blue, 0.25% dimethoate FF, 40% (mass concentration) sucrose aqueous solution. A8 Preparation of reaction components in reaction system A 8. 11 U TqDNA incubation medium
The volume of TagDNA synthase required in 25L reaction system is calculated according to formula (A.1): Voo
Wherein,
V—volume of TaqDNA synthase, in microliter (μL); D—concentration of TaqDNA synthase, in units per microliter (U/μL). A.8.21.5mmol/LMg2+
The volume of Mg+ added in 25uL reaction system is calculated according to formula (A2): 1, 5 X 25
Wherein,
V—volume of TaqDNA synthase, in microliter (μL);
D—concentration of Mg+, in millimole per liter (mmol/L). A, 8. 310 μmol/1, Primer
First centrifuge the primer, then dilute with water. The added volume is calculated according to formula (A, 3), stored in a 20℃ refrigerator, and diluted three times with water before each use, the final concentration is 10 μmol/L. OD2 × 33
Wherein, M in formula (A,3) is calculated according to formula (A.4): 1M = nA X 313.22+nG× 329.22+nCX 289.19+nT × 304.19 -61.97Wherein:
water volume, in microliters (L);
M—primer molecular weight, in grams per liter (/mL); ni—number of bases;
A—adenine;
guanine;
C. cytosine;
T—thymine.
Art Examination Literature
GB/T19564—2004
[1]J. Sambuke, EF Fritsch, T. Maniatis. Molecular cloning, Beijing, Science Press, 1996[2] Benjamin Levwin. Genes .NewYark:Oxford University Press,2000[3J Jain, A. , C. Apparanda, PL, Bhalla. Evaluation of genetic diversity and genome finger-printing of Pandorea (Bignoniaceae) by RAPD and inter-SSR PCR. Genome 42, 1999. 714~71[4 Kujimn. T. , T. Nagaoka, K. Noda, et al. Genetic linkage map nf ISSR and RAPD markersin Einkorn wheat in relation to that of RFLP markers, Theor. Appl. Genct. 96, 1998.37~45[5] Lanham. PG, R, M. Brennan. Genetic characterizatian af gooseherry (Ribes grossulariasubgenus Grossularia) germplasm using RAPD, ISSR and AFLP markers. J Hort Sci Biotech 74, 1999.361~366
[6] MeGregor. CE, C. A, Lambert, MM Greyling, et al. A cotnparative assessment ofDNA fingerprinting techniques (RAPD, ISSR, AF1.P and SSR) in tetraploid potato (Solanum tuberosutn L,)germplasm. Euphytica 113, 2000. 135~144[?] Naganka, T. , Y. Ogihara. Applicability of inter-simple sequence repeat polymorphisms inwheat for use as DNA markers in comparison to RFL.P and RAPD markers. Theor Appi Genet 94,1997.597~602
E8J Willinas JGK et al. DNA polymorphism anplificarion by arbitrary primers are useful as genetic markers.Nucleic AcidsRes,,1990.18(4):6531-6535KANiKAcaGenetic linkage map nf ISSR and RAPD markers in Einkorn wheat in relation to that of RFLP markers, Theor. Appl. Genct. 96, 1998.37~45[5] Lanham. PG, R, M. Brennan. Genetic characterizatian af gooseherry (Ribes grossulariasubgenus Grossularia) germplasm using RAPD, ISSR and AFLP markers. J Hort Sci Biotech 74,1999.361~366
[6] MeGregor. CE ,C. A, Lambert, MM Greyling, et al. A cotnparative assessment ofDNA fingerprinting techniques (RAPD, ISSR, AF1.P and SSR) in tetraploid potato (Solanum tuberosutn L,)germplasm. Euphytica 113, 2000. 135~144[?] Naganka, T. , Y. Ogihara. Applicability of inter-simple sequence repeat polymorphisms inwheat for use as DNA markers in comparison to RFL.P and RAPD markers. Theor Appi Genet 94,1997.597~602
E8J Willinas JGK et al. DNA polymorphism anplificarion by arbitrary primers are useful as genetic markers.Nucleic AcidsRes,,1990.18(4):6531-6535KANiKAcaGenetic linkage map nf ISSR and RAPD markers in Einkorn wheat in relation to that of RFLP markers, Theor. Appl. Genct. 96, 1998.37~45[5] Lanham. PG, R, M. Brennan. Genetic characterizatian af gooseherry (Ribes grossulariasubgenus Grossularia) germplasm using RAPD, ISSR and AFLP markers. J Hort Sci Biotech 74,1999.361~366
[6] MeGregor. CE ,C. A, Lambert, MM Greyling, et al. A cotnparative assessment ofDNA fingerprinting techniques (RAPD, ISSR, AF1.P and SSR) in tetraploid potato (Solanum tuberosutn L,)germplasm. Euphytica 113, 2000. 135~144[?] Naganka, T. , Y. Ogihara. Applicability of inter-simple sequence repeat polymorphisms inwheat for use as DNA markers in comparison to RFL.P and RAPD markers. Theor Appi Genet 94,1997.597~602
E8J Willinas JGK et al. DNA polymorphism anplificarion by arbitrary primers are useful as genetic markers.Nucleic AcidsRes,,1990.18(4):6531-6535KANiKAca
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