CN100429309C - 100bp gradient ribonucleic acid molecular weight marker and its preparation - Google Patents
100bp gradient ribonucleic acid molecular weight marker and its preparation Download PDFInfo
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Abstract
The present invention relates to a 100 bp gradient ribonucleic acid (DNA) molecular weight marker and a preparation method thereof, which belongs to the technical field of molecular biological reagent preparation. The molecular weight marker is a mixture composed of 11 DNA strips, wherein the length of the 11 DNA strips respectively are 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1000 bp and 1200 bp, and the concentration of each DNA strip is 4 ng/ mu l. In the preparation method, plasmids PBR322 are used as template DNA, under the condition of the existence of Taq DNA polymerase, Taq DNA polymerase buffer solutions and four kinds of deoxyribonucleotides (dTTP, dATP, dCTP and dGTP), 11 pairs of specific primers carry out the polymerase chain reaction on a PCR instrument under the same amplification condition, simultaneously, 11 DNA segments are synthesized, and then purification and mixing are carried out. The present invention has the characteristic of fast, accurately and largely preparing a large amount of DNA molecules with different length for the DNA molecular weight marker according to a predetermined target.
Description
Technical field
The present invention is to use polymerase chain reaction (PCR) technology, and the dna molecular amount mark of preparation 100bp gradient belongs to molecular biology reagent preparation field.
Background technology
Synthesize in the genetic manipulation process that reaches variety of way at DNA, need monitor synthetic or the dna molecular of operating size variation, method commonly used is to carry out electrophoresis simultaneously by the DNA hybrid standard material with dna molecular to be measured and a kind of known molecular amount, and the extent of migration in electrophoresis process is judged the size of dna molecular to be measured by dna molecular more to be measured and dna molecular reference material.Therefore, the standard DNA that needs some known molecular amounts of preparation.The method of preparation standard DNA has multiple:
Chemical synthesis
Method by chemical reaction is with 4 kinds of different deoxyribonucleotides: VITAMIN B4 (dATP), guanine (dGTP), cytosine(Cyt) (dCTP), thymus pyrimidine (dTTP) combines, the dna fragmentation of synthetic target sizes, and this method operation is very loaded down with trivial details, efficient is extremely low, synthetic DNA length is subjected to serious restriction, but the length of general synthetic DNA is no more than 400bp, and the dna fragmentation of macromolecule can't directly synthesize with chemical synthesis.
The fragment connection method
With the dna molecular of the more synthetic small molecular weights of chemical synthesis, with the enzymatic ligation small molecule DNA is progressively coupled together one by one more earlier, form the dna molecular of different sizes.The dna molecular that can prepare all size theoretically, but operation is very loaded down with trivial details, can't realize mass preparation.
Enzymolysis process
With one or both restriction enzymes to phage DNA (as φ χ 174), plasmid DNA (PBR322) is cut, produce the dna fragmentation of different sizes, as the molecular weight sign, the shortcoming of this method is the restriction that dna fragmentation size that enzyme is cut generation is subjected to used Restriction Enzyme kind, the preparation of dna molecular and purge process more complicated, cost height and productive rate are low.
The polymerase chain reaction method
The archaeal dna polymerase chain reaction has been widely used in the preparation of gene clone and dna fragmentation.Through consulting domestic patent documentation, still finding no with plasmid PBR322 is template, designs 11 pairs of Auele Specific Primers, and by polymerase chain reaction, the report of the 100bp gradient dna molecular amount mark of forming is with in preparation by 11 DNA.
Summary of the invention
The objective of the invention is to provide the method for a kind of dna molecular amount mark and quick preparation thereof based on the principle of polymerase chain reaction.The product of the present invention's preparation is used for molecular biology experiment the variation of dna molecular size is monitored as the molecular weight object of reference.
Technical scheme of the present invention:
100bp gradient dna molecular amount mark of the present invention is by 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1000bp, 1200bp, the mixtures formed of totally 11 DNA bands, be dissolved in the damping fluid (TE solution) of Tutofusin tris and disodium ethylene diamine tetraacetate composition, the final concentration of every DNA band is 4ng/ μ l.When carrying out the DNA electrophoresis, get 3-5 μ l molecular weight marker and sample to be detected and carry out electrophoresis simultaneously, electrophoresis is after for some time, molecular weight marker can on sepharose, produce 11 clearly, the DNA band that varies in size of molecular weight.The preparation of this molecular weight marker realizes by following technology, comprising: template DNA (plasmid PBR322), 11 pairs of Auele Specific Primers, the Taq archaeal dna polymerase, Taq dna polymerase buffer liquid, 4 kinds of deoxyribonucleotide (dTTP, dATP, dCTP, dGTP), deionized water, PCR instrument, sepharose, the pyridine of bromination second, bromjophenol blue, 0.25ml centrifuge tube, gel electrophoresis groove and electrophoresis power, ultra-violet lamp, ultraviolet spectrophotometer.Get the centrifuge tube of 11 aseptic 0.25ml, in each centrifuge tube, template DNA is mixed with a pair of Auele Specific Primer of corresponding DNA band, under the condition that Taq archaeal dna polymerase, Taq dna polymerase buffer liquid, 4 kinds of thymus nucleic acid existence are arranged, on the PCR instrument, under same pcr amplification condition, 11 dna fragmentations are carried out PCR amplification simultaneously, after 30 circulations, finish amplified reaction.The sample that takes a morsel from each reaction tubes carries out electrophoresis at the sepharose that contains the pyridine of bromination second, after electrophoresis finishes, observes the size and the purity of synthetic dna fragmentation on UV-lamp.Meet target sizes at definite amplified production, and after having the specificity of height, use phenol: chloroform is 1: 1 a mixed-solvent extraction, removes protein composition wherein; With ethanol precipitation deposit D NA molecule, dry sediment.With the DNA of the TE solution dissolution precipitation of certain volume, measure the concentration of dna solution with ultraviolet spectrophotometer.Is the mixed of 4ng/ μ l with 11 DNA bands by every final concentration, promptly can be made into the dna molecular amount mark of 100bp gradient.
Polymerase chain reaction principle: in Eppendorf tube, add an amount of damping fluid, the template DNA of trace, 4 kinds of deoxyribonucleotides (dNTP) solution, TaqDNA polysaccharase and a pair of Auele Specific Primer are with above-mentioned solution heating, make template DNA (as 95 ℃) sex change at high temperature, two strands is unwind into strand; Reduce solution temperature then, make synthetic primer (as 37 ℃) and the complementary annealing of template DNA at low temperatures, form the two strands of part; Solution temperature rises to middle temperature (as 72 ℃) again, under the effect of TaqDNA polysaccharase, is raw material with dNTP, to be combined in primer on the single-stranded template DNA is synthetic starting point, press the matching request of template DNA chain base, synthetic its complementary strand, such duplex molecule just becomes two duplex molecules.Change temperature of reaction so repeatedly in order, promptly high-temperature denatured, low-temperature annealing and middle temperature are extended synthetic, and such three temperature changes are a circulation, 1 times of every DNA number increase that makes the special section in the reaction tubes through once circulating.General sample finally makes primary template DNA quantity be increased to millions of times through about 30 times cyclic amplification.Certain dna molecular of mass production at short notice.
One, material:
1 plasmid PBR322 is general plasmid, is loop-like superhelix DNA, and total length is 4361bp, and its nucleotide sequence is as follows, and this plasmid is kept among the intestinal bacteria Ecoli JM109, available from U.S. PROMEGA company.
ttctcatgtt?tgacagctta?tcatcgataa?gctttaatgc?ggtagtttat?cacagttaaa 60
ttgctaacgc?agtcaggcac?cgtgtatgaa?atctaacaat?gcgctcatcg?tcatcctcgg 120
caccgtcacc?ctggatgctg?taggcatagg?cttggttatg?ccggtactgc?cgggcctctt 180
gcgggatat?c?gtccattccg?acagcatcgc?cagtcactat?ggcgtgctgc?tagcgctata 240
tgcgttgatg?caatttctat?gcgcacccgt?tctcggagca?ctgtccgacc?gctttggccg 300
ccgcccagtc?ctgctcgctt?cgctacttgg?agccactatc?gactacgcga?tcatggcgac 360
cacacccgtc?ctgtggatcc?tctacgccgg?acgcatcgtg?gccggcatca?ccggcgccac 420
aggtgcggtt?gctggcgcct?atatcgccga?catcaccgat?ggggaagatc?gggctcgcca 480
cttcgggctc?atgagcgctt?gtttcggcgt?gggtatggtg?gcaggccccg?tggccggggg 540
actgttgggc?gccatctcct?tgcatgcacc?attccttgcg?gcggcggtgc?tcaacggcct 600
caacctacta?ctgggctgct?tcctaatgca?ggagtcgcat?aagggagagc?gtcgaccgat 660
gcccttgaga?gccttcaacc?cagtcagctc?cttccggtgg?gcgcggggca?tgactatcgt 720
cgccgcactt?atgactgtct?tctttatcat?gcaactcgta?ggacaggtgc?cggcagcgct 780
ctgggtcatt?ttcggcgagg?accgctttcg?ctggagcgcg?acgatgatcg?gcctgtcgct 840
tgcggtattc?ggaatcttgc?acgccctcgc?tcaagccttc?gtcactggtc?ccgccaccaa 900
acgtttcggc?gagaagcagg?ccattatcgc?cggcatggcg?gccgacgcgc?tgggctacgt 960
cttgctggcg?ttcgcgacgc?gaggctggat?ggccttcccc?attatgattc?ttctcgcttc 1020
cggcggcatc?gggatgcccg?cgttgcaggc?catgctgtcc?aggcaggtag?atgacgacca 1080
tcagggacag?cttcaaggat?cgctcgcggc?tcttaccagc?ctaacttcga?tcactggacc 1140
gctgatcgtc?acggcgattt?atgccgcctc?ggcgagcaca?tggaacgggt?tggcatggat 1200
tgtaggcgcc?gccctatacc?ttgtctgcct?ccccgcgttg?cgtcgcggtg?catggagccg 1260
ggccacctcg?acctgaatgg?aagccggcgg?cacctcgcta?acggattcac?cactccaaga 1320
attggagcca?atcaattctt?gcggagaact?gtgaatgcgc?aaaccaaccc?ttggcagaac 1380
atatccatcg?cgtccgccat?ctccagcagc?cgcacgcggc?gcatctcggg?cagcgttggg 1440
tcctggccac?gggtgcgcat?gatcgtgctc?ctgtcgttga?ggacccggct?aggctggcgg 1500
ggttgcctta?ctggttagca?gaatgaatca?ccgatacgcg?agcgaacgtg?aagcgactgc 1560
tgctgcaaaa?cgtctgcgac?ctgagcaaca?acatgaatgg?tcttcggttt?ccgtgtttcg 1620
taaagtctgg?aaacgcggaa?gtcagcgccc?tgcaccatta?tgttccggat?ctgcatcgca 1680
ggatgctgct?ggctaccctg?tggaacacct?acatctgtat?taacgaagcg?ctggcattga 1740
ccctgagtga?tttttctctg?gtcccgccgc?atccataccg?ccagttgttt?accctcacaa 1800
cgttccagta?accgggcatg?ttcatcatca?gtaacccgta?tcgtgagcat?cctctctcgt 1860
ttcatcggta?tcattacccc?catgaacaga?aatccccctt?acacggaggc?atcagtgacc 1920
aaacaggaaa?aaaccgccct?taacatggcc?cgctttatca?gaagccagac?attaacgctt 1980
ctggagaaac?tcaacgagct?ggacgcggat?gaacaggcag?acatctgtga?atcgcttcac 2040
gaccacgctg?atgagcttta?ccgcagctgc?ctcgcgcgtt?tcggtgatga?cggtgaaaac 2100
ctctgacaca?tgcagctccc?ggagacggtc?acagcttgtc?tgtaagcgga?tgccgggagc 2160
agacaagccc?gtcagggcgc?gtcagcgggt?gttggcgggt?gtcggggcgc?agccatgacc 2220
cagtcacgta?gcgatagcgg?agtgtatact?ggcttaacta?tgcggcatca?gagcagattg 2280
tactgagagt?gcaccatatg?cggtgtgaaa?taccgcacag?atgcgtaagg?agaaaatacc 2340
gcatcaggcg?ctcttccgct?tcctcgctca?ctgactcgct?gcgctcggtc?gttcggctgc 2400
ggcgagcggt?atcagctcac?tcaaaggcgg?taatacggtt?atccacagaa?tcaggggata 2460
acgcaggaaa?gaacatgtga?gcaaaaggcc?agcaaaaggc?caggaaccgt?aaaaaggccg 2520
cgttgctggc?gtttttccat?aggctccgcc?cccctgacga?gcatcacaaa?aatcgacgct 2580
caagtcagag?gtggcgaaac?ccgacaggac?tataaagata?ccaggcgttt?ccccctggaa 2640
gctccctcgt?gcgctctcct?gttccgaccc?tgccgcttac?cggatacctg?tccgcctttc 2700
tcccttcggg?aagcgtggcg?ctttctcata?gctcacgctg?taggtatctc?agttcggtgt 2760
aggtcgttcg?ctccaagctg?ggctgtgtgc?acgaaccccc?cgttcagccc?gaccgctgcg 2820
ccttatccgg?taactatcgt?cttgagtcca?acccggtaag?acacgactta?tcgccactgg 2880
cagcagccac?tggtaacagg?attagcagag?cgaggtatgt?aggcggtgct?acagagttct 2940
tgaagtggtg?gcctaactac?ggctacacta?gaaggacagt?atttggtatc?tgcgctctgc 3000
tgaagccagt?taccttcgga?aaaagagttg?gtagctcttg?atccggcaaa?caaaccaccg 3060
ctggtagcgg?tggttttttt?gtttgcaagc?agcagattac?gcgcagaaaa?aaaggatctc 3120
aagaagatcc?tttgatcttt?tctacggggt?ctgacgctca?gtggaacgaa?aactcacgtt 3180
aagggatttt?ggtcatgaga?ttatcaaaaa?ggatcttcac?ctagatcctt?ttaaattaaa 3240
aatgaagttt?taaatcaatc?taaagtatat?atgagtaaac?ttggtctgac?agttaccaat 3300
gcttaatcag?tgaggcacct?atctcagcga?tctgtctatt?tcgttcatcc?atagttgcct 3360
gactccccgt?cgtgtagata?actacgatac?gggagggctt?accatctggc?cccagtgctg 3420
caatgatacc?gcgagaccca?cgctcaccgg?ctccagattt?atcagcaata?aaccagccag 3480
ccggaagggc?cgagcgcaga?agtggtcctg?caactttatc?cgcctccatc?cagtctatta 3540
attgttgccg?ggaagctaga?gtaagtagtt?cgccagttaa?tagtttgcgc?aacgttgttg 3600
ccattgctgc?aggcatcgtg?gtgtcacgct?cgtcgtttgg?tatggcttca?ttcagctccg 3660
gttcccaacg?atcaaggcga?gttacatgat?cccccatgtt?gtgcaaaaaa?gcggttagct 3720
ccttcggtcc?tccgatcgtt?gtcagaagta?agttggccgc?agtgttatca?ctcatggtta 3780
tggcagcact?gcataattct?cttactgtca?tgccatccgt?aagatgcttt?tctgtgactg 3840
gtgagtactc?aaccaagtca?ttctgagaat?agtgtatgcg?gcgaccgagt?tgctcttgcc 3900
cggcgtcaac?acgggataat?accgcgccac?atagcagaac?tttaaaagtg?ctcatcattg 3960
gaaaacgttc?ttcggggcga?aaactctcaa?ggatcttacc?gctgttgaga?tccagttcga 4020
tgtaacccac?tcgtgcaccc?aactgatctt?cagcatcttt?tactttcacc?agcgtttctg 4080
ggtgagcaaa?aacaggaagg?caaaatgccg?caaaaaaggg?aataagggcg?acacggaaat 2140
gttgaatact?catactcttc?ctttttcaat?attattgaag?catttatcag?ggttattgtc 4200
tcatgagcgg?atacatattt?gaatgtattt?agaaaaataa?acaaataggg?gttccgcgca 4260
catttccccg?aaaagtgcca?cctgacgtct?aagaaaccat?tattatcatg?acattaacct 4320
ataaaaatag?gcgtatcacg?aggccctttc?gtcttcaaga?a 4361
2 reagent
Intestinal bacteria (Ecoli JM109);
The plasmid DNA purification kit, Promega company produces;
Taq archaeal dna polymerase (1.5U/ μ l), Taq dna polymerase buffer liquid, magnificent biotechnology company limited produces;
4 * dNTP, by dTTP, dATP, dGTP, four kinds of monodeoxy ribonucleotides of dCTP are formed, and every kind concentration is 2.0mM;
Dna molecular amount mark is available from magnificent biotechnology company limited;
Penbritin,
Agarose,
The pyridine of bromination second, stock solution are 10mg/ml,
Bromjophenol blue,
Tutofusin tris (Tris),
Disodium ethylene diamine tetraacetate (EDTA-Na
2),
Re-distilled phenol,
Chloroform,
70% cold ethanol,
Dehydrated alcohol,
Peptone,
Yeast extract,
NaCl，
Agar powder,
Deionized water.
3 equipment
Micropipet,
The PCR instrument,
The Ultraluminescence lamp,
Ultraviolet spectrophotometer,
The low-temperature and high-speed whizzer,
Electrophoresis apparatus,
Incubator,
The constant temperature shaking table,
The vibration vortex mixer.
Two, reagent preparation
LB meat soup: peptone 1.0g
NaCl 1.0g
Yeast extract 0.5g
Transfer pH value to 7.2-7.4, add deionized water to 100ml.Autoclaving.
LB flat board: peptone 1.0g
NaCl 1.0g
Yeast extract 0.5g
Agar powder 1.2g
Transfer pH value to 7.2-7.4, add deionized water to 100ml.Autoclaving is cooled to 55 ℃ and paves ware.
TE damping fluid: Tris 10mM (PH 8.0)
EDTA-Na
2 1mM
Tbe buffer liquid: Tris-boric acid 0.045M (PH8.0)
EDTA-Na
2 0.001M
Gel load sample damping fluid: bromjophenol blue 0.25%
Sucrose 40% (w/v)
1% sepharose: in the Erlenmeyer flask of a 250ml, add 100ml tbe buffer liquid, 1.0g agarose, behind the mixing on microwave oven or electric furnace heating for dissolving, treat that agarose melts postcooling to 60 ℃ fully, adding bromination second pyridine to final concentration is 0.5 μ g/ml, records gel then in being inserted with the gel groove of comb, can use after the cooling.
Three, design of primers and synthetic
100bp: forward primer: 5 '-CTGAGCAACAACATGAAT-3 ' 1581-1598
Reverse primer: 5 '-CGATGCAGATCCGGAACA-3 ' 1678-1661
200bp: forward primer: 5 '-CTGAGCAACAACATGAAT-3 ' 1581-1598
Reverse primer: 5 '-GTATGGATGCGGCGGGACCA-3 ' 1778-1759
300bp: forward primer: 5 '-CTGAGCAACAACATGAAT-3 ' 1581-1598
Reverse primer: 5 '-GGTAATGATACCGATGAAACGA-3 ' 1878-1857
400bp: forward primer: 5 '-CTGAGCAACAACATGAAT-3 ' 1581-1598
Reverse primer: 5 '-GCGTTAATGTCTGGCTT-3 ' 1978-1962
500bp: forward primer: 5 '-CTGAGCAACAACATGAAT-3 ' 1581-1598
Reverse primer: 5 '-CGCGCGAGGCAGCT-3 ' 2078-2065
600bp: forward primer: 5 '-CTCCCTCGTG CGCTCTCCTGTTCC-3 ' 2642-2665
Reverse primer: 5 '-TTAATTTAAAAGGATCTAGGTG-3 ' 3239-3218
700bp: forward primer: 5 '-CCGTGTATGAAATCTAACAAT-3 ' 80-100
Reverse primer: 5 '-GCTGCCGGCACCTGTCCTACG-3 ' 777-757
800bp: forward primer: 5 '-CTGAGCAACAACATGAAT-3 ' 1581-1598
Reverse primer: 5 '-CGAGTCAGTGAGCGAGGA-3 ' 2378-2361
900bp: forward primer: 5 '-CTGAGCAACAACATGAAT-3 ' 1581-1598
Reverse primer: 5 '-ACATGTTCTTTCCTGCGTT-3 ' 2478-2460
1000bp: forward primer: 5 '-CTGAGCAACAACATGAAT-3 ' 1581-1598
Reverse primer: 5 '-CGTCGATTTTTGTGATGCT-3 ' 2578-2560
1200bp: forward primer: 5 '-CTGAGCAACAACATGAAT-3 ' 1581-1598
Reverse primer: 5 '-GCTTGGAGCGAACGACCTAC-3 ' 2778-2759
Primer is synthetic synthetic by the rich inferior biological products in Shanghai company limited, and the dry powder sample primer after synthesizing is dissolved into the solution that concentration is 12.5 μ M with deionized water.
Description of drawings
Fig. 1 pcr amplification is 1.100bp as a result, 2.200bp, 3.300bp, 4.400bp, 5.500bp, 6.600bp, 7.700bp, 8.800bp, 9.900bp, 10.1000bp, 11.1200bp.
The electrophoretogram of Figure 21 00bp gradient DNA mark material object.
The pcr amplification result of diagram 1. bacterium colonies 1 of Fig. 3 embodiment 2, the 2. pcr amplification result of bacterium colony 2,3, dna molecular amount sign.
Embodiment
The preparation method of 100bp gradient dna molecular amount mark specifically comprises the steps.
The preparation of 1 plasmid PBR322DNA
The microbionation that will contain plasmid PBR322 (contains penbritin 100 μ g/ml) in 3ml LB broth culture, 37 ℃ of shaking culture are spent the night.With the centrifugal collection of 12000g bacterium, use plasmid DNA purification kit purifying PBR322 plasmid again, the process that plasmid DNA is extracted is undertaken by the specification sheets of test kit.
2.DNA the preparation-polymerase chain reaction of band
Get the PCR thin-walled centrifuge tube of 11 0.25ml, put on 100bp respectively, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1000bp, the sign of 1200bp by following scheme, adds all ingredients that polymerase chain reaction is used in each pipe:
10 * Taq dna polymerase buffer liquid, 10 μ l
4×dNTP 8μl
PBR322?DNA 1μl(50ng)
Deionized water 75 μ l
After building lid, mixing boils sex change 10min in 100 ℃ of water-baths, and the cooling back is of short duration centrifugal, collects the water of condensation on the tube wall, and places on ice, adds the Taq archaeal dna polymerase (3U) of 2 μ l, and final reaction volume is 100 μ l.Reaction tubes is inserted in the hole slot of PCR instrument (PE2400) and carries out DNA cloning, amplification condition is set by following temperature:
94 ℃ of 10sec of sex change
50 ℃ of 20sec anneal
Extend 72 ℃ of 50sec
Cycle index is 30, last 72 ℃ of insulation 7min, termination reaction.
The evaluation of 3 polymeric enzyme reaction products
Sepharose is put into electrophoresis chamber, add electrophoretic buffer (TBE), make gel be dipped in about 3-5mm under the liquid level.Each amplified production is got 5 μ l mix, add in the gel pore with 2 μ l gel load sample damping fluids.Voltage with 1-5V/cm carries out electrophoresis, and sample is moved to negative electrode, stops electrophoresis when tetrabromophenol sulfonphthalein moves on to 2/3rds places of gel.Gel is placed on the ultraviolet lamp, observes amplification.Result such as Fig. 1.
4.DNA purifying and concentration determination
Each is contained in the centrifuge tube of the clean aseptic 1.5ml of the sample transfer to of unique DNA band, the re-distilled phenol that adds 1 times of volume, the chloroform of 1 times of volume, mixing 30sec on the vibration vortex mixer, again with the centrifugal 10min of the speed of 12000g, carefully the aqueous phase liquid that the upper strata is contained DNA is transferred in another clean centrifuge tube, add isopyknic re-distilled phenol more respectively, chloroform, behind the vibration mixing, centrifugal with same speed, supernatant liquor is transferred in another clean centrifuge tube, the chloroform that adds 2 times of volumes again, extracting is once again for identical method, at last supernatant liquor is transferred in the new clean centrifuge tube, the cold ethanol (30 ℃) that adds 2 times of volumes, put upside down mixing, place-80 ℃ of refrigerators to precipitate 15min, or precipitate 2h in-30 ℃ of refrigerators, again with the speed of 12000g at 4 ℃ of centrifugal 15min, outwell supernatant liquor, cold ethanol with 70% is washing precipitation and tube wall gently, carefully outwells ethanol, will have the sedimentary centrifuge tube of DNA to be placed on dry 10-20min under the room temperature, till beginning to bleach to the DNA throw out, with 10-50 μ l TE solution dissolution precipitation.
Get dna solution 2 μ l and mix, do 50 times of dilutions with 98 μ l TE solution.On ultraviolet spectrophotometer, measure the absorbance A value of solution at wavelength 280nm and 260nm place.Calculate the actual concentrations of DNA in the solution.
DNA concentration=A value * 50mg/ml * 50 (extension rate), [1A
OD280=50mg/ml]
The purity of DNA: A
260/ A
280=1.8-2.0
5.100bp the structure of gradient dna molecular amount mark
The total amount of setting every 100bp gradient dna molecular amount mark is 100 μ l, the final concentration of each dna fragmentation is 4ng/ul, each consumption is 5 μ l, and wherein the amount of 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1000bp, 1200bpDNA band is 20ng.According to every kind of dna fragmentation solution open beginning concentration, according to above setting requirement that 11 bar segment are admixed together, form the dna molecular amount mark of 100bp gradient, as Fig. 2.
Embodiment 2: be used to identify the positive colony bacterium colony
Transform dull and stereotyped bacterium colony 1 and the bacterium colony 2 gone up with the transfering loop picking, be suspended in the deionized water boiling lysis respectively.Be cracked into template with this, use gene-specific primer to carry out pcr amplification, get each 10 μ l of amplified production of 5 μ l molecular weight marker of the present invention and bacterium colony 1, bacterium colony 2, carry out agarose gel electrophoresis, electrophoresis finishes, observations under UV-light.The result shows, the amplified production of bacterium colony 1 produces the unique DNA band of molecular weight at 700-800bp, similar to the target gene size of being cloned, the positive clone's bacterium colony of preliminary this bacterium colony of proof, and the amplified production of bacterium colony 2 without any the DNA band occur, prove the negative clone's bacterium colony of this bacterium colony.As Fig. 3.
Claims (2)
1. 100bp gradient dna molecular amount mark, it is characterized in that being with the mixture of forming by 11 DNA, the final concentration of every DNA band is 4ng/ μ L, article 11, DNA band is to be template DNA with plasmid PBR322, with 11 pairs of Auele Specific Primers, through pcr amplification, the length of gained DNA band is respectively: 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1000bp, 1200bp;
The dna sequence dna of 11 pairs of primers is as follows:
100bp: forward primer: 5 '-CTGAGCAACAACATGAAT-3 ' 1581-1598
Reverse primer: 5 '-CGATGCAGATCCGGAACA-3 ' 1678-1661
200bp: forward primer: 5 '-CTGAGCAACAACATGAAT-3 ' 1581-1598
Reverse primer: 5 '-GTATGGATGCGGCGGGACCA-3 ' 1778-1759
300bp: forward primer: 5 '-CTGAGCAACAACATGAAT-3 ' 1581-1598
Reverse primer: 5 '-GGTAATGATACCGATGAAACGA-3 ' 1878-1857
400bp: forward primer: 5 '-CTGAGCAACAACATGAAT-3 ' 1581-1598
Reverse primer: 5 '-GCGTTAATGTCTGGCTT-3 ' 1978-1962
500bp: forward primer: 5 '-CTGAGCAACAACATGAAT-3 ' 1581-1598
Reverse primer: 5 '-CGCGCGAGGCAGCT-3 ' 2078-2065
600bp: forward primer: 5 '-CTCCCTCGTG CGCTCTCCTGTTCC-3 ' 2642-2665
Reverse primer: 5 '-TTAATTTAAAAGGATCTAGGTG-3 ' 3239-3218
700bp: forward primer: 5 '-CCGTGTATGAAATCTAACAAT-3 ' 80-100
Reverse primer: 5 '-GCTGCCGGCACCTGTCCTACG-3 ' 777-757
800bp: forward primer: 5 '-CTGAGCAACAACATGAAT-3 ' 1581-1598
Reverse primer: 5 '-CGAGTCAGTGAGCGAGGA-3 ' 2378-2361
900bp: forward primer: 5 '-CTGAGCAACAACATGAAT-3 ' 1581-1598
Reverse primer: 5 '-ACATGTTCTTTCCTGCGTT-3 ' 2478-2460
1000bp: forward primer: 5 '-CTGAGCAACAACATGAAT-3 ' 1581-1598
Reverse primer: 5 '-CGTCGATTTTTGTGATGCT-3 ' 2578-2560
1200bp: forward primer: 5 '-CTGAGCAACAACATGAAT-3 ' 1581-1598
Reverse primer: 5 '-GCTTGGAGCGAACGACCTAC-3 ' 2778-2759.
2. the preparation method of 100bp gradient dna molecular amount mark according to claim 1, it is characterized in that with plasmid PBR322 be template DNA, with 11 pairs of Auele Specific Primers, the Taq archaeal dna polymerase is being arranged, Taq dna polymerase buffer liquid, 4 kinds of deoxyribonucleotide dTTP, dATP, dCTP, under the condition that dGTP exists, on the PCR instrument, under same pcr amplification condition, carry out polymerase chain reaction, synthetic simultaneously 11 dna fragmentations, the length of gained DNA band is respectively: 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1000bp, 1200bp, purified again, mix:
(1) template DNA: the DNA that adopts plasmid PBR322;
(2) adopt 11 pairs of primers described in the claim 1, the concentration of every primer solution is 12.5 μ M;
(3) preparation-polymerase chain reaction of DNA band: the PCR thin-walled centrifuge tube of getting 11 0.25mL, put on the sign of 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1000bp, 1200bp respectively, by following scheme, in each pipe, add the reagent that polymerase chain reaction is used:
10 * TaqDNA polymerase buffer, 10 μ L
4×dNTP 8μL
Forward primer 2 μ L
Reverse primer 2 μ L
PBR322DNA 1 μ L is 50ng,
Deionized water 75 μ L
After building lid, mixing, in 100 ℃ of water-baths, boil sex change 10min, the cooling back is of short duration centrifugal, collects the water of condensation on the tube wall, and places on ice, the 3U Taq archaeal dna polymerase that adds 2 μ L, final volume is 100 μ L, reaction tubes is inserted in the hole slot of PCR instrument and carries out DNA cloning, by following temperature amplification condition is set:
94 ℃ of 10sec of sex change
50 ℃ of 20sec anneal
Extend 72 ℃ of 50sec
Cycle index is 30, last 72 ℃ of insulation 7min, termination reaction;
(4) evaluation of polymeric enzyme reaction product: sepharose is put into electrophoresis chamber, add electrophoretic buffer, make gel be dipped in 3-5mm under the liquid level, each amplified production is got 5 μ L to be mixed with 2 μ L gel load sample damping fluids, add in the gel pore, carry out electrophoresis, sample is moved to negative electrode with the voltage of 1-5V/cm, when bromjophenol blue moves on to 2/3rds places of gel, stop electrophoresis, on ultraviolet lamp, observe PCR degree of purity of production and output;
(5) DNA purifying and concentration determination: use phenol: chloroform is 1: 1 a mixed-solvent extraction, remove protein composition wherein, with ethanol precipitation deposit D NA molecule, dry sediment, the DNA of the buffer soln dissolution precipitation of forming with the Tutofusin tris of certain volume and disodium ethylene diamine tetraacetate measures the concentration of dna solution with ultraviolet spectrophotometer;
(6) structure of 100bp gradient dna molecular amount mark: with 100bp, 200bp, 300bp, 400bp, 500bp, 600bp, 700bp, 800bp, 900bp, 1000bp, 1200bp totally 11 DNA mixing, the final concentration that makes each band is 4ng/ μ L.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5714326A (en) * | 1991-01-24 | 1998-02-03 | Dawson; Elliott P. | Method for the multiplexed preparation of nucleic acid molecular weight markers and resultant products |
| US5824787A (en) * | 1993-12-03 | 1998-10-20 | Gensura Laboratories, Inc. | Polynucleotide sizing reagent |
| US6680378B1 (en) * | 1993-10-28 | 2004-01-20 | Invitrogen Corporation | Nucleic acid marker ladder for estimating mass |
| WO2004063322A2 (en) * | 2003-01-13 | 2004-07-29 | Seegene, Inc. | Dna size markers and method for preparing them |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5714326A (en) * | 1991-01-24 | 1998-02-03 | Dawson; Elliott P. | Method for the multiplexed preparation of nucleic acid molecular weight markers and resultant products |
| US6680378B1 (en) * | 1993-10-28 | 2004-01-20 | Invitrogen Corporation | Nucleic acid marker ladder for estimating mass |
| US5824787A (en) * | 1993-12-03 | 1998-10-20 | Gensura Laboratories, Inc. | Polynucleotide sizing reagent |
| WO2004063322A2 (en) * | 2003-01-13 | 2004-07-29 | Seegene, Inc. | Dna size markers and method for preparing them |
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