CN109504676A - A kind of DNA large fragment screening QIAquick Gel Extraction Kit and its application method - Google Patents
A kind of DNA large fragment screening QIAquick Gel Extraction Kit and its application method Download PDFInfo
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- CN109504676A CN109504676A CN201811457599.6A CN201811457599A CN109504676A CN 109504676 A CN109504676 A CN 109504676A CN 201811457599 A CN201811457599 A CN 201811457599A CN 109504676 A CN109504676 A CN 109504676A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
Abstract
The invention discloses a kind of DNA large fragment screening QIAquick Gel Extraction Kit and its application methods, including kit A and kit B, kit A electrophoresis liquid ingredient Tris 1240g, TAPS 1401.6g, EDTA 0.48g, seakem GOLD the Agarose 75g；Ingredient DNA eluent the NaI 40ml, sodium acetate 400ul, 100 μ l of magnetic bead of the kit B.The strict control of the present invention DNA large fragment screens the processing step of QIAquick Gel Extraction Kit and its application method, pass through the ingredient of strict control kit A and kit B, the big DNA fragmentation to 1Mb can be filtered out and substantially increase the DNA fragmentation screening for meeting large fragment sequencing, the DNA that 430kb-2Mb length can be recycled is suitble to the 3rd generation and the 4th generation gene sequencing of more long segment, can satisfy the technology of overlength sequencing.
Description
Technical field
The present invention relates to recycling gene chip technology field, specially a kind of DNA large fragment screens QIAquick Gel Extraction Kit, while this
Invention further relates to a kind of application method of DNA large fragment screening QIAquick Gel Extraction Kit.
Background technique
The short reading length read long sequencing technologies and generally produce 150-300bp of two traditional generation genes, in large-scale genome
On be difficult with traditional technology and be sequenced.The long sequencing of short reading can not cross over complete DNA repeat region, and what is be generated by it is existing
The assembling of high proportion genome shows corresponding repeat region and structure variation, and there is many incomplete gaps.It comes within 2018
The Christo Si Puluoka Keyes (Christos Proukakis) of University College London (UCL) Neurology Research Institute is situated between
Continued a kind of method using long sequencing GBA gene.This method has used the amplicon comprising all code areas and introne,
It is successfully sequenced to from 17 individual DNA samples including Parkinson's disease and high Xue Shi disease patient, Sang Ge
Method is sequenced or carries out the long targeting sequencing of short reading, all letters required for these methods will fail, omit variation or can not provide
Breath.With the development of sequencing technologies, the screening reagent box in view of current longest segment is 300kb, the at present screening of more long segment
QIAquick Gel Extraction Kit lacks, and leads to not complete more complete long segment sequencings.If glucocerebrosidase is about the gene of 8kb, it
Nearby there is the pseudogene of a 20kb；The spiral bladderwort of genome 61Mb, Paris polyphylla lily of 152Gb etc., longer mrna length
Sequencing, become to be highly desirable.Longest recycling genetic fragment is only less than in traditional DNA recycling and preparation
The length of 200Kb is much unable to satisfy the technology of overlength sequencing.
Summary of the invention
The purpose of the present invention is to provide a kind of DNA large fragment screening QIAquick Gel Extraction Kit and its application methods, on solving
State the problem of proposing in background technique.
To achieve the above object, the invention provides the following technical scheme: a kind of DNA large fragment screens QIAquick Gel Extraction Kit, packet
Kit A and kit B, kit A electrophoresis liquid ingredient Tris 1240g, TAPS 1401.6g, the EDTA 0.48g are included,
seakem GOLD Agarose 75g；The ingredient DNA eluent NaI 40ml of the kit B, sodium acetate 400ul, magnetic bead
100μl。
Preferably, the kit A totally 10 secondary response, SYBR 5u；Totally 10 secondary response, SYBR are the kit B
50ul。
The present invention also provides a kind of application methods of DNA large fragment screening QIAquick Gel Extraction Kit, comprising the following steps:
S1: the running gel bed of organic glass being cleaned, is dried, and is sealed the opening at both ends with adhesive tape, is placed on horizontal
On workbench, sample comb is plugged；
S2: Tris 124g, TAPS 140.16g, EDTA 0.48g is taken to add the dH2O of 1L to be placed at room temperature for spare every time；
S3: the preparation of Ago-Gel weighs agarose placement, then takes in step 2 and dissolve in liquid, sets micro-wave oven or boiling
It is heated to dissolving completely in water-bath, taking-up shakes up；
S4: liquid 50ml in step 2, seakem GOLD Agarose 75g is taken to dissolve in the dH2O of 950ml；
S5: the seakem GOLD Agarose solution for being cooled to 60 DEG C is gently poured on electrophoresis tank level board；
S6: after seakem GOLD Agarose gelling is solid, solution in step 2 is added in electrophoresis tank, then extracts comb
Son；
S7: mixed liquor is added in sample cell by DNA sample with micropipettor, and every slot adds 10-20 μ l, records sample
Point sample order and sample-adding amount；
S8: installing electrode cable, and loading wells one terminates cathode, and another termination is positive, opening power supply, tune voltage to 75V,
Electric pulse 16h-20h stops electric pulse after reaching the time；
S9: SYBR 5ul is added into developer solution, is put into gel shading and covers, places 30 minutes；
S10: segment agarose blob of viscose required for gel is quickly cut under blue light gel imager is taken out, is added
The NaI solution of 400ul, water-bath 10min；
S11: the 2.5mol/L sodium acetate and 400ul dehydrated alcohol of 40ul are added into step S10 solution, in -20 DEG C of items
15-30min is placed under part, is centrifuged 10min under the conditions of revolving speed is 10000rpm；
S12: precipitating pours into 70% ethyl alcohol with 70% ethanol wash again after twice, place magnetic bead 10ul in liquid and stand
2min gentle agitation 1 time, removes magnetic bead, leaves and takes liquid after circulation；
S13: dd H2O is added after air-drying with 70% ethanol wash 1 time in precipitating, and places under the conditions of 2-8 DEG C.
Preferably, the Ago-Gel amount prepared in the step S3 is 110ml.
Preferably, electric pulse uses model Part No.PPI0200 in the step S8.
Preferably, the concentration for the NaI solution being added in the step S10 is 6mol/L.
Preferably, the bath temperature condition in the step S10 is 50-60 DEG C.
Preferably, the cycle-index of the step S12 is 2 times.
Compared with prior art, the beneficial effects of the present invention are: the screening recycling examination of the strict control of the present invention DNA large fragment
The processing step of agent box and its application method can be filtered out greatly by the ingredient of strict control kit A and kit B to 1Mb
DNA fragmentation substantially increase meet large fragment sequencing DNA fragmentation screening, can recycle 430kb-2Mb length DNA be suitble to
3rd generation of more long segment and the 4th generation gene sequencing can satisfy the technology of overlength sequencing.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, below in conjunction with specific embodiment, to this
Invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, not
For limiting the present invention.
Embodiment 1
A kind of DNA large fragment screens QIAquick Gel Extraction Kit, including kit A and kit B, the kit A electrophoresis liquid at
Divide Tris 1240g, TAPS 1401.6g, EDTA 0.48g, seakem GOLD Agarose75g；The ingredient of the kit B
DNA eluent NaI 40ml, sodium acetate 400ul, 100 μ l of magnetic bead.
Specifically, the kit A totally 10 secondary response, SYBR 5u；Totally 10 secondary response, SYBR are the kit B
50ul。
The present invention also provides a kind of application methods of DNA large fragment screening QIAquick Gel Extraction Kit, comprising the following steps:
S1: the running gel bed of organic glass being cleaned, is dried, and is sealed the opening at both ends with adhesive tape, is placed on horizontal
On workbench, sample comb is plugged；
S2: Tris 124g, TAPS 140.16g, EDTA 0.48g is taken to add the dH2O of 1L to be placed at room temperature for spare every time；
S3: the preparation of Ago-Gel weighs agarose placement, then takes in step 2 and dissolve in liquid, sets micro-wave oven or boiling
It is heated to dissolving completely in water-bath, taking-up shakes up；
S4: liquid 50ml in step 2, seakem GOLD Agarose 75g is taken to dissolve in the dH2O of 950ml；
S5: the seakem GOLD Agarose solution for being cooled to 60 DEG C is gently poured on electrophoresis tank level board；
S6: after seakem GOLD Agarose gelling is solid, solution in step 2 is added in electrophoresis tank, then extracts comb
Son；
S7: mixed liquor is added in sample cell by DNA sample with micropipettor, and every slot adds 10 μ l, records the point of sample
Sample order and sample-adding amount；
S8: installing electrode cable, and loading wells one terminates cathode, and another termination is positive, opening power supply, tune voltage to 75V,
Electric pulse 16h stops electric pulse after reaching the time；
S9: SYBR 5ul is added into developer solution, is put into gel shading and covers, places 30 minutes；
S10: segment agarose blob of viscose required for gel is quickly cut under blue light gel imager is taken out, is added
The NaI solution of 400ul, water-bath 10min；
S11: the 2.5mol/L sodium acetate and 400ul dehydrated alcohol of 40ul are added into step S10 solution, in -20 DEG C of items
15min is placed under part, is centrifuged 10min under the conditions of revolving speed is 10000rpm；
S12: precipitating pours into 70% ethyl alcohol with 70% ethanol wash again after twice, place magnetic bead 10ul in liquid and stand
2min gentle agitation 1 time, removes magnetic bead, leaves and takes liquid after circulation；
S13: dd H2O is added after air-drying with 70% ethanol wash 1 time in precipitating, and places under the conditions of 2 DEG C.
Specifically, the Ago-Gel amount prepared in the step S3 is 110ml.
Specifically, electric pulse uses model Part No.PPI0200 in the step S8.
Specifically, the concentration for the NaI solution being added in the step S10 is 6mol/L.
Specifically, the bath temperature condition in the step S10 is 50 DEG C.
Specifically, the cycle-index of the step S12 is 2 times.
Embodiment 2
A kind of DNA large fragment screens QIAquick Gel Extraction Kit, including kit A and kit B, the kit A electrophoresis liquid at
Divide Tris 1240g, TAPS 1401.6g, EDTA 0.48g, seakem GOLD Agarose75g；The ingredient of the kit B
DNA eluent NaI 40ml, sodium acetate 400ul, 100 μ l of magnetic bead.
Specifically, the kit A totally 10 secondary response, SYBR 5u；Totally 10 secondary response, SYBR are the kit B
50ul。
The present invention also provides a kind of application methods of DNA large fragment screening QIAquick Gel Extraction Kit, comprising the following steps:
S1: the running gel bed of organic glass being cleaned, is dried, and is sealed the opening at both ends with adhesive tape, is placed on horizontal
On workbench, sample comb is plugged；
S2: Tris 124g, TAPS 140.16g, EDTA 0.48g is taken to add the dH2O of 1L to be placed at room temperature for spare every time；
S3: the preparation of Ago-Gel weighs agarose placement, then takes in step 2 and dissolve in liquid, sets micro-wave oven or boiling
It is heated to dissolving completely in water-bath, taking-up shakes up；
S4: liquid 50ml in step 2, seakem GOLD Agarose 75g is taken to dissolve in the dH2O of 950ml；
S5: the seakem GOLD Agarose solution for being cooled to 60 DEG C is gently poured on electrophoresis tank level board；
S6: after seakem GOLD Agarose gelling is solid, solution in step 2 is added in electrophoresis tank, then extracts comb
Son；
S7: mixed liquor is added in sample cell by DNA sample with micropipettor, and every slot adds 15 μ l, records the point of sample
Sample order and sample-adding amount；
S8: installing electrode cable, and loading wells one terminates cathode, and another termination is positive, opening power supply, tune voltage to 75V,
Electric pulse 18h stops electric pulse after reaching the time；
S9: SYBR 5ul is added into developer solution, is put into gel shading and covers, places 30 minutes；
S10: segment agarose blob of viscose required for gel is quickly cut under blue light gel imager is taken out, is added
The NaI solution of 400ul, water-bath 10min；
S11: the 2.5mol/L sodium acetate and 400ul dehydrated alcohol of 40ul are added into step S10 solution, in -20 DEG C of items
22.5min is placed under part, is centrifuged 10min under the conditions of revolving speed is 10000rpm；
S12: precipitating pours into 70% ethyl alcohol with 70% ethanol wash again after twice, place magnetic bead 10ul in liquid and stand
2min gentle agitation 1 time, removes magnetic bead, leaves and takes liquid after circulation；
S13: dd H2O is added after air-drying with 70% ethanol wash 1 time in precipitating, and places under the conditions of 5 DEG C.
Specifically, the Ago-Gel amount prepared in the step S3 is 110ml.
Specifically, electric pulse uses model Part No.PPI0200 in the step S8.
Specifically, the concentration for the NaI solution being added in the step S10 is 6mol/L.
Specifically, the bath temperature condition in the step S10 is 55 DEG C.
Specifically, the cycle-index of the step S12 is 2 times.
Embodiment 3
A kind of DNA large fragment screens QIAquick Gel Extraction Kit, including kit A and kit B, the kit A electrophoresis liquid at
Divide Tris 1240g, TAPS 1401.6g, EDTA 0.48g, seakem GOLD Agarose75g；The ingredient of the kit B
DNA eluent NaI 40ml, sodium acetate 400ul, 100 μ l of magnetic bead.
Specifically, the kit A totally 10 secondary response, SYBR 5u；Totally 10 secondary response, SYBR are the kit B
50ul。
The present invention also provides a kind of application methods of DNA large fragment screening QIAquick Gel Extraction Kit, comprising the following steps:
S1: the running gel bed of organic glass being cleaned, is dried, and is sealed the opening at both ends with adhesive tape, is placed on horizontal
On workbench, sample comb is plugged；
S2: Tris 124g, TAPS 140.16g, EDTA 0.48g is taken to add the dH2O of 1L to be placed at room temperature for spare every time；
S3: the preparation of Ago-Gel weighs agarose placement, then takes in step 2 and dissolve in liquid, sets micro-wave oven or boiling
It is heated to dissolving completely in water-bath, taking-up shakes up；
S4: liquid 50ml in step 2, seakem GOLD Agarose 75g is taken to dissolve in the dH2O of 950ml；
S5: the seakem GOLD Agarose solution for being cooled to 60 DEG C is gently poured on electrophoresis tank level board；
S6: after seakem GOLD Agarose gelling is solid, solution in step 2 is added in electrophoresis tank, then extracts comb
Son；
S7: mixed liquor is added in sample cell by DNA sample with micropipettor, and every slot adds 20 μ l, records the point of sample
Sample order and sample-adding amount；
S8: installing electrode cable, and loading wells one terminates cathode, and another termination is positive, opening power supply, tune voltage to 75V,
Electric pulse 20h stops electric pulse after reaching the time；
S9: SYBR 5ul is added into developer solution, is put into gel shading and covers, places 30 minutes；
S10: segment agarose blob of viscose required for gel is quickly cut under blue light gel imager is taken out, is added
The NaI solution of 400ul, water-bath 10min；
S11: the 2.5mol/L sodium acetate and 400ul dehydrated alcohol of 40ul are added into step S10 solution, in -20 DEG C of items
30min is placed under part, is centrifuged 10min under the conditions of revolving speed is 10000rpm；
S12: precipitating pours into 70% ethyl alcohol with 70% ethanol wash again after twice, place magnetic bead 10ul in liquid and stand
2min gentle agitation 1 time, removes magnetic bead, leaves and takes liquid after circulation；
S13: dd H2O is added after air-drying with 70% ethanol wash 1 time in precipitating, and places under the conditions of 8 DEG C.
Specifically, the Ago-Gel amount prepared in the step S3 is 110ml.
Specifically, electric pulse uses model Part No.PPI0200 in the step S8.
Specifically, the concentration for the NaI solution being added in the step S10 is 6mol/L.
Specifically, the bath temperature condition in the step S10 is 60 DEG C.
Specifically, the cycle-index of the step S12 is 2 times.
In a kind of DNA large fragment screening QIAquick Gel Extraction Kit and its application method proposed by the present invention, the experimental data are shown in the following table
1:
In summary: the strict control of the present invention DNA large fragment screens the technique step of QIAquick Gel Extraction Kit and its application method
Suddenly, by the ingredient of strict control kit A and kit B, the big DNA fragmentation to 1Mb can be filtered out and substantially increased meet
The DNA fragmentation screening of large fragment sequencing, the DNA that can recycle 430kb-2Mb length are suitble to the 3rd generation and the 4th generation of more long segment
Gene sequencing can satisfy the technology of overlength sequencing.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Anyone skilled in the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Claims (8)
1. a kind of DNA large fragment screens QIAquick Gel Extraction Kit, including kit A and kit B, it is characterised in that: the kit A
Electrophoresis liquid ingredient Tris 1240g, TAPS 1401.6g, EDTA 0.48g, seakem GOLD Agarose 75g；The reagent
Ingredient DNA eluent the NaI 40ml, sodium acetate 400ul, 100 μ l of magnetic bead of box B.
2. a kind of DNA large fragment according to claim 1 screens QIAquick Gel Extraction Kit, it is characterised in that: the kit A is total
10 secondary responses, SYBR 5u；The kit B totally 10 secondary response, SYBR 50ul.
3. the application method of DNA large fragment screening QIAquick Gel Extraction Kit, special described in a kind of -2 any one according to claim 1
Sign is: the following steps are included:
S1: the running gel bed of organic glass being cleaned, is dried, and is sealed the opening at both ends with adhesive tape, is placed on horizontal work
On platform, sample comb is plugged；
S2: Tris 124g, TAPS 140.16g, EDTA 0.48g is taken to add the dH2O of 1L to be placed at room temperature for spare every time；
S3: the preparation of Ago-Gel weighs agarose placement, then takes in step 2 and dissolve in liquid, sets micro-wave oven or boiling water bath
In be heated to dissolving completely, taking-up shakes up；
S4: liquid 50ml in step 2, seakem GOLD Agarose 75g is taken to dissolve in the dH2O of 950ml；
S5: the seakem GOLD Agarose solution for being cooled to 60 DEG C is gently poured on electrophoresis tank level board；
S6: after seakem GOLD Agarose gelling is solid, solution in step 2 is added in electrophoresis tank, then extracts comb；
S7: mixed liquor is added in sample cell by DNA sample with micropipettor, and every slot adds 10-20 μ l, records the point sample of sample
Order and sample-adding amount；
S8: installing electrode cable, and loading wells one terminates cathode, and another termination anode opens power supply, adjusts voltage to 75V, electric arteries and veins
16h-20h is rushed, stops electric pulse after reaching the time；
S9: SYBR 5ul is added into developer solution, is put into gel shading and covers, places 30 minutes；
S10: segment agarose blob of viscose required for gel is quickly cut under blue light gel imager is taken out, is added 400ul's
NaI solution, water-bath 10min；
S11: the 2.5mol/L sodium acetate and 400ul dehydrated alcohol of 40ul are added into step S10 solution, under the conditions of -20 DEG C
15-30min is placed, is centrifuged 10min under the conditions of revolving speed is 10000rpm；
S12: precipitating pours into 70% ethyl alcohol with 70% ethanol wash again after twice, place magnetic bead 10ul in liquid and stand 2min,
Gentle agitation 1 time, magnetic bead is removed after circulation, leaves and takes liquid；
S13: dd H2O is added after air-drying with 70% ethanol wash 1 time in precipitating, and places under the conditions of 2-8 DEG C.
4. a kind of application method of DNA large fragment screening QIAquick Gel Extraction Kit according to claim 1, it is characterised in that: institute
Stating the Ago-Gel amount prepared in step S3 is 110ml.
5. a kind of application method of DNA large fragment screening QIAquick Gel Extraction Kit according to claim 1, it is characterised in that: institute
It states electric pulse in step S8 and uses model Part No.PPI0200.
6. a kind of application method of DNA large fragment screening QIAquick Gel Extraction Kit according to claim 1, it is characterised in that: institute
The concentration for stating the NaI solution being added in step S10 is 6mol/L.
7. a kind of application method of DNA large fragment screening QIAquick Gel Extraction Kit according to claim 1, it is characterised in that: institute
Stating the bath temperature condition in step S10 is 50-60 DEG C.
8. a kind of application method of DNA large fragment screening QIAquick Gel Extraction Kit according to claim 1, it is characterised in that: institute
The cycle-index for stating step S12 is 2 times.
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Cited By (1)
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| CN112391380A (en) * | 2019-12-04 | 2021-02-23 | 武汉未来组生物科技有限公司 | Ultralong DNA purification method and reagent thereof |
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Application publication date: 20190322 |