CN101575601A - Super plasmid of DNA molecular weight standard for producing even 200 bp gradient and preparation method thereof - Google Patents
Super plasmid of DNA molecular weight standard for producing even 200 bp gradient and preparation method thereof Download PDFInfo
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- CN101575601A CN101575601A CNA2008100157799A CN200810015779A CN101575601A CN 101575601 A CN101575601 A CN 101575601A CN A2008100157799 A CNA2008100157799 A CN A2008100157799A CN 200810015779 A CN200810015779 A CN 200810015779A CN 101575601 A CN101575601 A CN 101575601A
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Abstract
The invention relates to a super plasmid for producing a DNA molecular weight standard with an even 200 bp gradient and a preparation method thereof. The tailing activity of a Taq DNA polymerase and the selective gel recycle of a connecting product are utilized to construct a brand-new plasmid pYO 7200 for producing the DNA molecular weight standard with the oven 200 bp gradient. The plasmid is a brand-new super DNA molecular weight standard plasmid and comprises seven sections of 400 bp, 600 bp, 800 bp, 1000 bp, 1400 bp, 2000 bp and 3000 bp, wherein the section of 400 bp is double copies so as to enhance the fluorescence intensity of the 400 bp after electrophoresis during dyeing observation; the plasmid is processed by enzyme digestion by EcoR I to obtain an even 200 bp gradient DNA molecular weight standard gradient comprising seven sections, and the densities (masses) of the seven DNA bands have a certain proportional relation of 4:3:4:5:7:10:15. The invention overcomes the defect that the size of a DNA section produced by a traditional enzyme digestion method is limited by used restricted enzyme types, is easy to produce on a large scale and has high production rate.
Description
Technical field
The present invention relates to molecular biology reagent preparation and genetically engineered Application Areas, be a kind of super plasmid that makes up by genetic engineering means and preparation method thereof specifically, this super plasmid is used to use the method for digestion with restriction enzyme to prepare even 200 bp gradient dna molecular amount standard.
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 thing (molecular weight standard, DNA marker) with dna molecular to be measured and a kind of known molecular amount, and the migration distance in electrophoresis process is judged the size of DNA sample molecule by dna molecular more to be measured and dna molecular standard.Therefore, the standard DNA fragment mixture that needs some known molecular amounts of preparation.
The existing several different methods of the method for preparation standard DNA in the prior art:
Chemical synthesis: the method by chemical reaction is examined picric acids with 4 kinds of different ribodesoses: VITAMIN B4 (dATP), guanine (dGTP), cytosine(Cyt) dCTP), thymus pyrimidine (dTTP) combines, the dna fragmentation of synthetic target sizes.This method operation is very loaded down with trivial details, and efficient is extremely low, and synthetic DNA length is subjected to serious restriction, but the length of general synthetic DNA is no more than 400hp, the dna fragmentation of macromolecule can't directly synthesize with chemical synthesis, and cost is too high, thereby the preparation of the routine of inapplicable dna molecular amount.
Fragment connection method: the dna molecular (as 100bp) that obtains small molecular weight earlier with PCR or synthetic method, with the enzymatic ligation small molecule DNA is linked in sequence one by one again, form different sizes dna moleculars (100bp, 200bp, 300bp, 400bp ...).Say the dna molecular that can prepare all size on this theoretical method, but operation is very loaded down with trivial details, reproducibility is poor, can't realize mass preparation.
The polymerase chain reaction method: the archaeal dna polymerase chain reaction has been widely used in the preparation as gene clone and the DNA sheet.Be widely used for preparing dna molecular amount standard substance at present both at home and abroad.But this method wastes time and energy, and its throughput is limited.
Enzyme cutting method: cut with the plasmid of one or both restriction enzymes combination, produce the dna fragmentations of different sizes, with as dna molecular amount standard to phage DNA or artificial design.The advantage of this method is a large amount of easily preparations, the productive rate height.But the shortcoming of this method is also very outstanding: the dna fragmentation size that enzyme is cut generation is subjected to the restriction of used Restriction Enzyme kind, and its restraining factors are design levels of artificial constructed plasmid.
For a long time, molecular biology reagent and genetically engineered Application Areas, need a kind of high-caliber artificial constructed plasmid design, cut the restriction that the dna fragmentation size of generation is subjected to used Restriction Enzyme kind to overcome enzyme, to produce even 200 bp gradient dna molecular amount standard fast, accurately, in large quantities.
Summary of the invention
Through secular laboratory study, the present invention has exactly satisfied the demand, and its purpose is: provide a kind of that make up, brand-new by genetic engineering means, can produce the super plasmid of 200bp even number gradient dna molecular amount standard fast, accurately, in large quantities by the enzyme blanking method.
The present invention can further purpose be: the preparation method that a kind of super plasmid of the 200bp of production even number gradient dna molecular amount standard is provided.
To achieve these goals, the present invention has adopted following technical scheme:
Method by PCR is increased respectively 400bp, 600bp, 800bp, 1000bp, several bar segment of 1400bp, 2000bp of template with Lambda DNA, wherein 1400bp and 1000bp increase by the pfu archaeal dna polymerase, and 400bp-A, 400bp-B, 600bp, 800bp increase with the Taq archaeal dna polymerase respectively.Utilize the tailing activity of Taq archaeal dna polymerase that 1400bp and 1000bp two bar segment are added T then.Utilize the A/T complementary principle system that in centrifuge tube, connects again, utilize the T4 ligase enzyme to make 600bp, 800bp, 1000bp connect into the unit of a 600-800-1000, same 400bp-A, 400bp-B, 1400bp connect into the unit of 400A-1400-400B.By T/A clone test kit, said two units is cloned into formation intermediate carrier T681 and T4144 in the T carrier respectively then.
Cut by Pst I enzyme the 400A-1400-400B unit is cut out from T4144, and utilize nucleic acid purification to reclaim test kit and reclaim and purifying.While T681 carrier is also cut holoenzyme by Pst I enzyme and cuts, and utilizes calf intestine alkaline phosphatase (CIAP) to carry out the carrier dephosphorization treatment, to prevent that carrier is from connecting.Set up suitable linked system, the 400-1400-400 unit is connected the into T681 carrier gained intermediate carrier called after T6814144 of dephosphorization treatment.By the technological line close with said process, utilize PCR method from Lambda DNA, to increase and obtain the fragment of a 2000bp, and be cloned in the T carrier, cut out again with restriction endonuclease Sph I then, be connected into same Sph I enzyme and cut T6814144 with the CIAP dephosphorization treatment, obtain super plasmid, called after pYE9600.
The super plasmid pYE9600 that is obtained is a kind of brand-new super dna molecular amount standard plasmid.Wherein contain 400bp, 600bp, 800bp, 1000bp, 1400bp, 2000bp six bar segment, wherein 400bp is two copies, the fluorescence intensity when observing to strengthen it.Plasmid can obtain a 200bp even number dna molecular amount normal gradients of being made up of 7 bar segment after EcoR I enzyme is cut.Had certain ratio row relation simultaneously between the concentration (quality) of each bar DNA band (4: 3: 4: 5: 7: 10: 15).
Beneficial effect of the present invention is: utilize sophisticated production technique and genetically engineered to make up a kind of artificial super plasmid, this plasmid is a kind of brand-new dna molecular amount standard plasmid, this plasmid is cut preparation 200bp even number gradient dna molecular amount standard, the dna fragmentation size that has overcome traditional enzyme cutting method generation is subjected to the restriction of used Restriction Enzyme kind, a large amount of easily preparations, the productive rate height.Realized purpose of the present invention.
Description of drawings
Fig. 1 is a super plasmid pYE9600 collection of illustrative plates of producing even 200 bp gradient dna molecular amount standard.
Embodiment
Produce the super plasmid preparation method of even 200 bp gradient dna molecular amount standard, specifically comprise the steps:
1. increase respectively 400bp, 600bp, 800bp, 1000bp, several bar segment of 1400bp, 2000bp of the method by PCR, wherein 1400bp and 1000bp increase by the pfu archaeal dna polymerase, and 400bp, 600bp, 800bp increase with the Taq archaeal dna polymerase respectively.
2. utilize the tailing activity of Taq archaeal dna polymerase that 1400bp and 1000bp two bar segment are added T.
3. utilize the A/T complementation system that connects in centrifuge tube, make 600bp, 800bp, 1000bp connect into the unit of 600-800-1000,400bp and 1400bp connect into the unit of 400-1400-400.
4. by T/A clone test kit, two unit of above-mentioned 600-800-1000,400-1400-400 are cloned into formation intermediate carrier T681 and T4144 in the T carrier respectively.
5. cut by Pst I enzyme the 400-1400-400 unit is cut out from T4144, and reclaim.While T681 carrier is also cut holoenzyme by Pst I enzyme and cuts, and utilizes CIAP to carry out the carrier dephosphorization treatment, to prevent that carrier is from connecting.
6. the T681 carrier gained intermediate carrier T6814144 that the 400-1400-400 unit is connected into above-mentioned dephosphorization treatment.
7.CIAP dephosphorization treatment T6814144.
8.2000bp be cloned in the T carrier, cut out again with restriction endonuclease Sph I then, be connected into same Sph I enzyme and cut T6814144 with the CIAP dephosphorization treatment, obtain plasmid pYE9600.
This plasmid is a kind of brand-new super dna molecular amount standard plasmid.Wherein contain 400bp, 600bp, 800bp, 1000bp, 1400bp, 2000bp, 3000bp seven bar segment, wherein 400bp is two copies, the fluorescence intensity when observing to strengthen it.Plasmid can obtain a 200bp even number dna molecular amount normal gradients of being made up of 7 bar segment after EcoR I enzyme is cut.Had certain ratio row relation simultaneously between the concentration (quality) of each bar DNA band (4: 3: 4: 5: 7: 10: 15).
Claims (4)
1. produce the super plasmid of even 200 bp gradient dna molecular amount standard, this plasmid is a kind of brand-new super dna molecular amount standard plasmid, it is characterized in that: wherein contain 400bp, 600bp, 800bp, 1000bp, 1400bp, 2000bp, 3000bp seven bar segment; 400bp is two copies; After cutting, EcoR I enzyme can obtain a 200bp even number dna molecular amount normal gradients of forming by 7 bar segment; Had certain ratio row relation simultaneously between the concentration (quality) of each bar DNA band (4: 3: 4: 5: 7: 10: 15).
2. the preparation method of the super plasmid of production even 200 bp gradient dna molecular amount standard according to claim 1, comprise method by PCR increase respectively 400bp, 600bp, 800bp, 1000bp, several bar segment of 1400bp, 2000bp, it is characterized in that:
(1) 400bp, 600bp, 800bp are increased by general T aq archaeal dna polymerase respectively;
(2) 1400bp, 1000bp are increased by the pfu archaeal dna polymerase respectively;
(3) utilize the tailing activity of Taq archaeal dna polymerase that 1400bp and 1000bp two bar segment are added T;
(4) utilize the A/T complementation system that connects in centrifuge tube, make 600bp, 800bp, 1000bp connect into the unit of 600-800-1000,400bp and 1400bp connect into the unit of 400-1400-400;
(5) by T/A clone test kit, two unit of above-mentioned 600-800-1000,400-1400-400 are cloned into formation intermediate carrier T681 and T4144 in the T carrier respectively;
(6) cut by Pst I enzyme the 400-1400-400 unit is cut out from T4144, and reclaim; Simultaneously the T681 carrier is also by Pst I complete degestion, and utilizes CIAP to carry out the carrier dephosphorization treatment;
(7) the T681 carrier that the 400-1400-400 unit is connected into above-mentioned dephosphorization treatment obtains intermediate carrier T6814144;
(8) with the 2000bp fragment cloning in the T carrier, cut out again with restriction endonuclease Sph I then, be connected into same Sph I enzyme and cut T6814144 with the CIAP dephosphorization treatment, obtain super plasmid pYE9600.
3. the super plasmid preparation method of production even 200 bp gradient dna molecular amount standard according to claim 2, it is characterized in that: described 400bp, 600bp, 800bp increase by polysaccharase respectively, specifically are meant with the Taq archaeal dna polymerase and increase.
4. the super plasmid preparation method of production even 200 bp gradient dna molecular amount standard according to claim 2, it is characterized in that: described 1400bp, 1000bp increase by polysaccharase respectively, specifically are meant with the pfu archaeal dna polymerase and increase.
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| CNA2008100157799A CN101575601A (en) | 2008-05-07 | 2008-05-07 | Super plasmid of DNA molecular weight standard for producing even 200 bp gradient and preparation method thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101880661A (en) * | 2010-06-11 | 2010-11-10 | 济南大学 | 200bp DNA ladder template p222 based on PCR (Polymerase Chain Reaction) amplification and preparation system thereof |
| CN102304508A (en) * | 2011-07-29 | 2012-01-04 | 上海捷瑞生物工程有限公司 | Method for preparing DL2000 DNA molecular weight marker as well as product and applications thereof |
| CN102337284A (en) * | 2011-09-30 | 2012-02-01 | 生工生物工程(上海)有限公司 | Plasmid for preparing DNA Marker, and construction method and application thereof |
| CN106906231A (en) * | 2017-04-10 | 2017-06-30 | 济南大学 | A kind of even number DNA molecular amount standard and preparation method thereof |
-
2008
- 2008-05-07 CN CNA2008100157799A patent/CN101575601A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101880661A (en) * | 2010-06-11 | 2010-11-10 | 济南大学 | 200bp DNA ladder template p222 based on PCR (Polymerase Chain Reaction) amplification and preparation system thereof |
| CN102304508A (en) * | 2011-07-29 | 2012-01-04 | 上海捷瑞生物工程有限公司 | Method for preparing DL2000 DNA molecular weight marker as well as product and applications thereof |
| CN102304508B (en) * | 2011-07-29 | 2013-05-01 | 上海捷瑞生物工程有限公司 | Method for preparing DL2000 DNA molecular weight marker as well as product and applications thereof |
| CN102337284A (en) * | 2011-09-30 | 2012-02-01 | 生工生物工程(上海)有限公司 | Plasmid for preparing DNA Marker, and construction method and application thereof |
| CN106906231A (en) * | 2017-04-10 | 2017-06-30 | 济南大学 | A kind of even number DNA molecular amount standard and preparation method thereof |
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