CN103995039B - A kind of SSR amplified production native polyacrylamide gel electrophoresis method and device - Google Patents
A kind of SSR amplified production native polyacrylamide gel electrophoresis method and device Download PDFInfo
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- CN103995039B CN103995039B CN201410234799.0A CN201410234799A CN103995039B CN 103995039 B CN103995039 B CN 103995039B CN 201410234799 A CN201410234799 A CN 201410234799A CN 103995039 B CN103995039 B CN 103995039B
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Abstract
The invention discloses a kind of SSR amplified production native polyacrylamide gel electrophoresis method and devices, the present apparatus is with three pieces of glass plates using the sandwich combination of 3 folders 2, separation gel is poured between two interlayers formed between three pieces of glass plates simultaneously, two pieces of separation hectographs can be made in an encapsulating.Since the concave size of two pieces of glass plates with square breach is different, the height for forming the loading wells of latter two hectograph of gel is different, and when point sample, mistake was less likely to occur, and avoiding should be o'clock on the point to another hectograph of the sample mistake on a hectograph.An encapsulating of the invention can make two pieces of hectographs simultaneously, compare 30% or more working efficiency raising with the conventional one piece of glue that can only once fill with glue, encapsulating process.In subsequent fixation, dyeing, fixing, developing process, the hectograph quantity of single treatment increases compared with conventional method, can totally save the dosages such as electrode buffer, developer solution, dyeing liquor, fixer 30% or so.
Description
Technical field
The invention belongs to technological field of biochemistry, are related to a kind of SSR amplified production non-denaturing polyacrylamide gel electricity
Swimming method and device.
Background technique
Gel electrophoresis technology is due to having many advantages, such as quick, easy and high-resolution, using very extensive.From separation with
Inorganic ions is analyzed to complicated biological polymeric compound, and all occupies important ground in radiochemistry and immunochemistry
Position；Using more common in terms of Biochemical Lab and biotechnology industry；Medical department also serves as clinical diagnosis.In all kinds of electrophoresis skills
In art, using gel electrophoresis, resolving power is made a living as highest in terms ofs separating enzyme analysis, protein, nucleic acid etc. especially
Major contribution has been made in object chemistry, the development of molecular biology.Encapsulating of existing method can only make 1 block of vertical panel gel electricity
Swim hectograph, 1 normal vertical plate gel electrophoresis slot at most can simultaneously the fast hectograph of electrophoresis 2.
Summary of the invention
It is an object of the invention to overcome defect existing for above-mentioned technology, a kind of SSR amplified production non denatured poly- third is provided
Acrylamide gel electrophoresis method and device can make 2 pieces of glue, 1 normal vertical plate gel electrophoresis slot using the present apparatus simultaneously
At most the fast hectograph of electrophoresis 4, electrophoresis tank utilization efficiency 1 times can be improved simultaneously, improve 30% or more working efficiency.Its particular technique
Scheme are as follows:
A kind of SSR amplified production native polyacrylamide gel electrophoresis method, comprising the following steps:
The preparation of 1 polyacrylamide gel:
1.1 cleaning glass plates, encapsulating graduated cylinder and beaker, glass plate gas is dry, and (glue glass plate encapsulating face must be clean
Otherwise no moisture is easy to produce bubble when encapsulating；Beaker, graduated cylinder and glass plate easily make if there is water since glue is immiscible with water
Glue cannot securely glue on a glass)；
1.2 are placed on flat mouth glass plate and recess glass plate on the supporter (column vitreum cushion block etc.) of desktop, give glass
Glass plate drips a small amount of 100% ethyl alcohol, uniformly cleans glue face with high-quality paper, gas is dry, and scrobicula glass plate first cleans one with alcohol
Face is overturn after gas is dry, then is wiped another side gas and done；
Two short sides of 1.3 flat mouth glass plates and recess glass plate uniformly smear appropriate vaseline；
First glass plate is placed on cushion block when making hectograph, then puts one respectively at the edge close to two short side
Adhesive tape, adhesive tape short side and does not have jagged glass plate bottom edge to put together, and the short side of long side glass plate is parallel, with hand by adhesive tape by firm
Gu putting scrobicula glass plate on two adhesive tape, a glue is similarly then being put respectively close to glass plate short side edge
Then item is placed on deep concave glass plate in adhesive tape again with hand by jail；It is required that the notch of two pieces of recess glass plates is in same direction
On, the other three side of three pieces of glass plates stacks neatly；Two interlayers are formed between three pieces of glass plates in this way, are finally used
Clip is fixed；
1.4 match glue, encapsulating:
Following mother liquor is mixed in quantity and is uniformly stored in 4 DEG C, is finished in general one week,
Size and thickness for selected glue measure the appropriate above mixed liquor and pour into clean beaker, are added appropriate 10%
APS and TEMED, shakes up, and under laid-flat status, the separation gel prepared is poured into two interlayers from recess side, slowly insertion with
The comb of glue consistency of thickness, while avoiding generating bubble under comb teeth, it is solid wait be gelled；
2 electrophoresis
Appropriate vaseline is uniformly smeared on 2.1 rubber strips seeped in the case where electrophoresis tank prevents buffer, for preventing upper slot slow
Fliud flushing penetrates into lower slot, and appropriate 1x TBE electrophoretic buffer is first added toward upper and lower electrophoresis tank；
After 2.2 gellings are solid, comb taken out, and slightly rinsed at loading wells with water, glass plate is sideling put into electrophoresis tank,
It avoids glue bottom end and buffer joint from generating bubble, glass plate is fixed on electrophoresis tank with clip, buffer is added, makes
Upper slot buffer at least covers loading wells, and lower slot buffer at least with glue lower edge flush, is then rinsed with electrophoretic buffer
Loading wells；
2.3 5x the Loading dye, each sample spot 10-12 μ L of 2 μ L are added into 10 μ LPCR amplification sample；Finally
Point Marker DNA；
2.4 constant pressures or constant current electrophoresis, constant current；2 plates, electric current 100-120mA, voltage about 300-400V；4 plates, electric current 200-
240mA, voltage about 300-400V；But voltage should be kept constant, otherwise band is easily bent；
The colour developing of 3 silver stainings
3.1 when the first dyestuff is close to glue feather edge, and electrophoresis terminates, and the first dyestuff can also be made to run out of；Electrophoresis apparatus is closed, is moved
Clip is removed, glass plate is removed, two layers of glue is carefully removed from glass plate respectively, then starts silver staining processing, processing is being shaken
It is carried out on bed, the specific steps are as follows:
20 minutes are fixed in fixer (10% acetic acid) first, pure water rinsing is then spent 15 minutes, is put into dyeing liquor
It is dyed 30 minutes in 0.2% silver nitrate, with pure water rinsing 10-20 seconds, is put into developer solution and suitably develops, be then put into fixing solution
It is fixed 5 minutes in 10% acetic acid, takes a picture after fixing；
It should be noted that item in 3.2 silver stainings:
3.2.1 the quality of water influences dyeing effect greatly, ultrapure water or distilled water generally to be used, if water contains pollution
Object, glue may not develop or only show upper strap portion；And lower part blank；
3.2.2 the purity of sodium carbonate is also critically important in developer solution, it is proposed that uses natrium carbonicum calcinatum；
3.2.3 since temperature is excessively high, develop too fast, so generally storing developer solution for the ease of controlling developing process
In 4 DEG C, the used time is taken out；Developer solution is easy crystallization in 4 DEG C long-term place, and is generally prepared using first 1-2 days, developer solution is prepared
Method is as follows: weighing Na first2CO3100 grams of (natrium carbonicum calcinatum) then measures 160 microlitres of 50mg/ml sodium thiosulfate, then
2 milliliters of formaldehyde are added, constant volume is to 1000 milliliters；(note: Ying Yu -20 DEG C of 50mg/ml hypo solution preservations)
3.2.4 a few drop glycerol are added in the fixing solution 10%, as last developer solution；
3.2.5 each step should all gently shake, and carry out generally on shaking table；
3.2.6 the washing time after silver staining in deionized water requires very stringent, generally at 10 seconds or so, should be less than
20 seconds, if it exceeds 20 seconds, Ying Chongxin contaminates 30 minutes again in 0.2% silver nitrate；
3.2.7 0.2% silver nitrate solution may be reused 3-4 times, and the silver staining time is appropriately extended every time；
3.2.8 because not easy to clean, dress silver nitrate, developer solution cannot be mixed with the basin of fixer and fixing solution；
3.2.9 after electrophoresis, after smearing the glass plate of vaseline, lath hot-water soak, vaseline is wiped with paper,
It is easier to clean when cleaning again, or is cleaned after impregnating with dish washing liquid；
4 photograph
With gel image analyser photograph or dry glue scanning.
Further preferably,
(1) Acr and Bis is never poison, is irritating to the skin effect, and when operation should wear gloves and mask；(2) glass
Plate surface answers smooth pieces, otherwise will cause in electrophoresis and generates bubble between gel slab and glass plate；(3) sample template comb teeth
It answers flat and smooth；(4) there cannot be a bubble when filling gel, in case electric current passes through when influencing electrophoresis；(5) it is sure not to destroy sample-adding groove
Bottom it is smooth, in order to avoid electrophoresis back zone band distort；(6) lath must be consistent with comb thickness, otherwise will generate in loading wells
Glue film influences point sample and last electrophoresis quality)；(7) glue should be chalcogenide glass with glass plate；The loading wells of (8) two hectographs
Height difference there was only 0.5cm, when point sample should be noted that avoid should sample mistake o'clock on a hectograph point to other one
On a hectograph；(9) due to simultaneously electrophoresis hectograph number it is more, it is different to distinguish Marker point on different swimming lanes
Hectograph；(10) constant pressure or constant current electrophoresis, constant current；2 offset plates of electrophoresis, electric current are set as 100-120mA, voltage about 300-400V；
4 offset plates of electrophoresis, electric current are set as 200-240mA, voltage about 300-400V；But voltage should be kept constant, otherwise band holds
It is flexible.
A kind of device for realizing SSR amplified production native polyacrylamide gel electrophoresis method of the present invention, packet
Include 3 pieces of glass plate, adhesive tape 4, comb 1,2, clip；
Three pieces of glass plates outer dimension used is long and wide all identical, and glass plate 2 is rectangle, having a size of 20cm × 17cm；Shallowly
Concave glass plate 3 is the notch for having a recessed 1.5cm × 17cm in a long side；Deep concave glass plate 4 have a recessed 2.0cm ×
The notch of 17cm, the thickness of glass plate are all 3mm；Adhesive tape length is 18cm, broadband 1cm, the glue that thickness makes as needed
Version thickness adjustment, generally 1mm；The thickness of comb is identical with the thickness of adhesive tape, the number of teeth have 15,20,40 it is not, according to experiment
It is required that selection.
First glass plate 2 is placed on cushion block when making hectograph, is then put respectively in two short side close to marginal position
One adhesive tape, adhesive tape short side and does not have jagged glass plate bottom edge to put together, and long side is parallel with the short side of glass plate, in two adhesive tape
Put scrobicula glass plate 3 above, then similarly putting an adhesive tape respectively close to 3 short side edge of glass plate, then again
Glass plate 4 is placed in adhesive tape.It is required that the notch of two pieces of recess glass plates is in the same direction, the other three side of three pieces of glass plates
It stacks neatly, is formed two interlayers between three pieces of glass plates in this way, is finally clipped in and is stacked respectively with two clips
Three pieces of glass plates two short side middle positions.Under laid-flat status, the separation gel prepared is poured into two folders from recess side
In layer, plugs comb and puts on the support frame after glue polymerization completely to be concentrated, clamp fixation with clip, lightly take out comb,
Loading wells is cleaned with distilled water immediately, so that it may form the hectograph with two pieces of separation gels.Other one is made in the same way
Fast hectograph is individually placed to the both sides of vertical slab electrophoresis groove, with clamp, toward the buffer chamber up and down of electrophoresis tank in electrode is added
Sample is clicked and entered sample well bottom with microsyringe, so that it may carry out electrophoresis by buffer.
Compared with prior art, the invention has the benefit that
An encapsulating of the invention can make two pieces of hectographs simultaneously, can only once fill one with glue, encapsulating process and routine
Block glue improves 30% or more compared to working efficiency.Electrophoresis tank is integrally placed to as one using 2 pieces of hectographs that the present apparatus is produced
Side, electrophoresis tank two sides can most 4 pieces of hectographs of electrophoresis, and use most 2 pieces of electrophoresis of conventional offset production method
The utilization efficiency of glue, electrophoresis tank is doubled.Simultaneously because the hectograph number of an electrophoresis tank electrophoresis increases one times, electrophoresis apparatus
Utilization efficiency also double, save 50% electric energy.The experiment carried out in two times is originally needed, it now once can be complete
At in the case where the limited amount of electrophoresis apparatus, needing to wait the time of electrophoresis to reduce half, accelerate test process.It is comprehensive
In production hectograph, the electrophoresis waiting time is reduced, cleans the quantity of glass plate, and one in fixation, dyeing, fixing, developing process
The hectograph quantity of secondary processing increases and the time of saving, general work efficiency can be improved 30% or more.
In terms of saving the energy, the present apparatus makes 4 pieces of hectographs and only needs 6 pieces of glass plates, and conventional method is with 8 pieces, brush glass
The time of glass plate and water consumption reduce 25%.It is compared with two pieces of offset plate methods of an electrophoresis tank electrophoresis of existing use, an electrophoresis
Slot can increase one times with the hectograph quantity of electrophoresis, and the electrode buffer needed reduces half.Due in subsequent fixation, dye
Color, fixing, in developing process, the hectograph quantity of single treatment increases compared with conventional method, can totally save electrode buffer, aobvious
The dosages such as shadow liquid, dyeing liquor, fixer 30% or so.
Detailed description of the invention
Fig. 1 is silver staining development step flow chart；
Fig. 2 is rectangular glass；
Fig. 3 is scrobicula glass plate；
Fig. 4 is deep concave glass plate；
Fig. 5 is adhesive tape；
Fig. 6 is comb.
Specific embodiment
Technical solution of the present invention is described in more detail in the following with reference to the drawings and specific embodiments.
A kind of SSR amplified production native polyacrylamide gel electrophoresis method, comprising the following steps:
1) prepared by polyacrylamide gel hectograph
1. cleaning glass plate, encapsulating graduated cylinder and beaker, glass plate gas is dry, and (glue is chalcogenide glass, encapsulating with glass plate
Face must clean no moisture, bubble is otherwise easy to produce when encapsulating；Beaker, graduated cylinder and glass plate are if there is water, due to glue and water
It is immiscible, easily prevent glue from securely gluing on a glass)；
2. flat mouth glass plate and recess glass plate are placed on the supporter (glass block etc.) of desktop, dripped to glass plate few
100%7 alcohol are measured, uniformly clean glue face with high-quality paper, gas is dry；
3. two short sides of flat mouth glass plate and recess glass plate uniformly smear appropriate vaseline, (vaseline is not allowed too much
Easy cleaning)；The lath for needing thickness is put, with hand otherwise two sides are easy leak adhesive by securely；Then (folder is clamped with clip
Sub-folder is among lath).
2) with glue by taking 8% non-denaturing polyacrylamide gel as an example, encapsulating Acr: Bis=37.5: 1)；
Following mother liquor is mixed in quantity and is uniformly stored in 4 DEG C, is finished in general one week.
Size and thickness for selected glue measure the appropriate above mixed liquor and pour into clean beaker, are added appropriate 10%
APS (catalyst) and TEMED (accelerator), shakes up, encapsulating, slowly the comb of insertion and glue consistency of thickness, to avoid under comb teeth
Bubble is generated, it is solid wait be gelled.
For the glass plate of 20cm x17cm, slat dimension is removed, glue is 16.5cm (width) x15.5cm (height) x1mm
(thickness),
It prepares as shown in table 1:
Table 1
3) electrophoresis
1. appropriate 1x TBE electrophoretic buffer is first added in buffering liquid groove above and below electrophoresis tank, the reusable 3- of buffer
4 times；
2. after gelling is solid, slowly taking out comb (being careful not to that comb teeth is made to deform or pull off comb teeth), slightly being rinsed with water
At loading wells, glass plate is sideling put into electrophoresis tank, glue bottom end and buffer joint is avoided to generate bubble, with clip by glass
Plate is fixed on electrophoresis tank, adds buffer, and slot buffer is made at least to cover loading wells, under lower slot buffer interface and glue
Edge flush, and do not had the lower edge of glue at least, then loading wells is rinsed with electrophoretic buffer；
3. the 5x Loading dye of 2 μ L is added into 10 μ LPCR amplification sample, and (this step can generally be completed in PCR amplification
After carry out, so that solution be made to be sufficiently mixed), each sample spot 10-12 μ L；Rearmost point Marker DNA；
4. constant pressure or constant current electrophoresis, constant current.2 plates, 100-120mA, voltage about 300-400V；4 plates, 200-240mA, voltage
About 300-400V；But voltage should be kept constant, otherwise band is easily bent；
4) silver staining develops the color
1. electrophoresis terminates when the first dyestuff is close to glue feather edge, the first dyestuff can also be made to run out of；Electrophoresis apparatus is closed, is removed
Clip removes glass plate, and glue is carefully removed from glass plate, then starts silver staining processing, and processing carries out on shaking table, has
Body step is as shown in Figure 1:
It should be noted that item in silver staining:
1. the quality of water influences dyeing effect greatly, ultrapure water or distilled water generally to be used, if water contains pollutant, glue
May not develop or only show upper strap portion；And lower part blank；
2. the purity of sodium carbonate is also critically important in developer solution, it is proposed that use natrium carbonicum calcinatum；
3. developing too fast since temperature is excessively high, so developer solution is generally stored in 4 for the ease of controlling developing process
DEG C, the used time takes out；Developer solution is easy crystallization in 4 DEG C long-term place, and is generally prepared using first 1-2 days, developer solution preparation side
Method is as shown in table 2:
Table 2
(note: Ying Yu -20 DEG C of 50mg/ml hypo solution preservations)
4. a few drop glycerol are added in the fixing solution 10%, last developer solution may be used as；
5. each step all should gentle agitation, carried out generally on shaking table；
6. the washing time requirement after silver staining in deionized water is very stringent, generally at 10 seconds or so, should be less than 20 seconds,
If it exceeds 20 seconds, Ying Chongxin contaminates 30 minutes again in 0.2% silver nitrate；
7. 0.2% silver nitrate solution may be reused 3-4 times, the silver staining time is appropriately extended every time；
8. dress silver nitrate, developer solution cannot be mixed with the basin of fixer and fixing solution because not easy to clean；
9. after electrophoresis, after smearing the glass plate of vaseline, lath hot-water soak, wiping vaseline with paper, then clearly
It is easier to clean when washing, or is cleaned after impregnating with dish washing liquid.
7 photograph
Photograph or dry glue scanning.
Preferably, (1) Acr and Bis is never poison, is irritating to the skin effect, and when operation should wear gloves and mask；
(2) glass pane surface answers smooth pieces, otherwise will cause in electrophoresis and generates bubble between gel slab and glass plate；(3) sample
Template comb teeth is answered flat and smooth；(4) there cannot be a bubble when filling gel, in case electric current passes through when influencing electrophoresis；(5) it not touch
Electrophoresis back zone band is caused to distort in order to avoid destroying the smooth of sample-adding notch board bottom in the bottom of hectograph；(6) lath and comb thickness must
Must be consistent, glue film otherwise will be generated in loading wells, influences point sample and last electrophoresis quality)；(7) due to simultaneously electrophoresis hectograph
Number is more, Marker point on different swimming lanes, to distinguish different hectographs.The height of the loading wells of (8) two hectographs
Difference only has 0.5cm, when point sample should be noted that avoid should sample mistake o'clock on a hectograph point to another hectograph
On；(9) glue should be chalcogenide glass with glass plate.
As shown in figures 2-6, a kind of realization SSR amplified production of the present invention native polyacrylamide gel electrophoresis side
The device of method, including 3 pieces of glass plate, adhesive tape 4, comb 1,2, clip.
Three pieces of glass plates outer dimension used is long and wide all identical.The size of rectangular glass is 20cm × 17cm scrobicula glass
Glass plate is the notch for having a recessed 1.5cm × 17cm in a long side；Deep concave glass plate has lacking for a recessed 2.0cm × 17cm
Mouthful, the thickness of glass plate is all 3mm.Adhesive tape length is 18cm, width 1cm, the hectograph thickness tune that thickness makes as needed
It is whole, generally 1mm.The thickness of comb is identical with the thickness of adhesive tape, the number of teeth have 15,20,40 it is not, selected according to requirement of experiment
It selects.
First rectangular glass is placed on two pieces of identical cushion blocks when making hectograph, then on the side close to two short side
Edge puts an adhesive tape respectively, adhesive tape short side and does not have jagged glass plate bottom edge to put together, and long side is parallel with the short side of glass plate,
Scrobicula glass plate is put on two adhesive tape, is then similarly putting a glue respectively close to scrobicula glass plate short side edge
Then item is placed on deep concave glass plate in adhesive tape again.It is required that the notch of two pieces of recess glass plates is in the same direction, three blocks of glass
The other three side of plate stacks neatly, is formed two interlayers between three pieces of glass plates in this way, is finally pressed from both sides respectively with clip
In two short side middle positions of the three pieces of glass plates stacked.Under laid-flat status, by the separation gel prepared from recess side
It pours into two interlayers, plugs comb and put on the support frame after glue polymerization completely to be concentrated, clamp fixation with clip, take out comb
Son cleans loading wells with distilled water immediately, so that it may form the hectograph with two pieces of separation gels.It makes in the same way in addition
One fast hectograph, is individually placed to the both sides of vertical slab electrophoresis groove, with clamp, toward the buffer chamber up and down of electrophoresis tank in electricity is added
Sample is clicked and entered sample well bottom with microsyringe, so that it may carry out electrophoresis by pole buffer.
Claims (3)
1. a kind of SSR amplified production native polyacrylamide gel electrophoresis method, which comprises the following steps:
The preparation of 1 polyacrylamide gel:
1.1 cleaning glass plates, encapsulating graduated cylinder and beaker, glass plate gas are dry；
1.2 are placed on flat mouth glass plate and recess glass plate on the supporter of desktop, drip a small amount of 100% ethyl alcohol to glass plate, use
High-quality paper uniformly cleans glue face, and gas is dry, and scrobicula glass plate first cleans one side with alcohol, overturns after gas is dry, then wipe another
Gas is dry on one side；
Two short sides of 1.3 flat mouth glass plates and recess glass plate uniformly smear appropriate vaseline；
First glass plate is placed on cushion block when making hectograph, then puts a glue respectively at the edge close to two short side
Item, adhesive tape short side and does not have jagged glass plate bottom edge to put together, and the short side of long side glass plate is parallel, with hand by adhesive tape by secured,
Scrobicula glass plate is put on two adhesive tape, is then similarly putting an adhesive tape respectively close to glass plate short side edge,
With hand by jail, then glass plate is placed in adhesive tape again；It is required that the notch of two pieces of recess glass plates is in the same direction, three pieces of glass
The other three side of glass plate stacks neatly；Two interlayers are formed between three pieces of glass plates in this way, are finally fixed with clip；
1.4 match glue, encapsulating:
Following mother liquor is mixed in quantity and is uniformly stored in 4 DEG C, is finished in one week,
Size and thickness for selected glue measure the appropriate above mixed liquor and pour into clean beaker, and appropriate 10%APS is added
And TEMED, it shakes up, under laid-flat status, the separation gel prepared is poured into two interlayers from recess side, slowly insertion and glue are thick
Consistent comb is spent, while avoiding generating bubble under comb teeth, it is solid wait be gelled；
2 electrophoresis
Appropriate vaseline is uniformly smeared on 2.1 rubber strips seeped in the case where electrophoresis tank prevents buffer, for preventing upper slot buffer
Lower slot is penetrated into, appropriate 1 × TBE electrophoretic buffer is first added toward upper and lower electrophoresis tank；
After 2.2 gellings are solid, comb taken out, and slightly rinsed at loading wells with water, glass plate is sideling put into electrophoresis tank, avoids glue
Bottom end and buffer joint generate bubble, and glass plate is fixed on electrophoresis tank with clip, adds buffer, keep slot slow
Fliud flushing at least covers loading wells, and lower slot buffer at least with glue lower edge flush, then rinses loading wells with electrophoretic buffer；
2.3 5x the Loading dye, each sample spot 10-12 μ L of 2 μ L are added into 10 μ LPCR amplification sample；Rearmost point
Marker DNA；
2.4 constant pressures or constant current electrophoresis, constant current；2 offset plates of electrophoresis, electric current are set as 100-120mA, voltage 300-400V；Electrophoresis 4
A offset plate, electric current are set as 200-240mA, voltage 300-400V；But voltage should be kept constant, otherwise band is easily bent；
The colour developing of 3 silver stainings
3.1 first dyestuffs close electrophoresis apparatus, electrophoresis terminates, removes clip, remove glass plate, by two layers of glue close to glue feather edge
It is carefully removed from glass plate respectively, then starts silver staining processing, processing carries out on shaking table, the specific steps are as follows:
20 minutes are fixed in 10% acetic acid fixer first, are then used pure water rinsing 15 minutes, are put into 0.2% nitric acid of dyeing liquor
It is dyed 30 minutes in silver, with pure water rinsing 10-20 seconds, is put into developer solution and suitably develops, be then put into 10% acetic acid of fixing solution
Middle fixing 5 minutes, takes a picture after fixing；
It should be noted that item in 3.2 silver stainings:
3.2.1 the quality of water influences dyeing effect greatly, to select ultrapure water or distilled water, if water contains pollutant, glue can
Can not develop or only show upper strap portion；And lower part blank；
3.2.2 the purity of sodium carbonate is also critically important in developer solution, uses natrium carbonicum calcinatum；
3.2.3 since temperature is excessively high, develop too fast, so developer solution is stored in 4 DEG C, is used for the ease of controlling developing process
When take out；Developer solution is easy crystallization in 4 DEG C long-term place, and is prepared using first 1-2 days, development liquid making method is as follows: first
100 grams of natrium carbonicum calcinatum are first weighed, then measures 160 microlitres of 50mg/ml sodium thiosulfate, is then added 2 milliliters of formaldehyde, constant volume
To 1000 milliliters；
3.2.4 a few drop glycerol are added in the fixing solution 10%, as last developer solution；
3.2.5 each step all gently shakes, and carries out on shaking table；
3.2.6 the washing time after silver staining in deionized water requires very stringent, and washing time should be less than 20 seconds, if it exceeds
20 seconds, Ying Chongxin contaminated 30 minutes again in 0.2% silver nitrate；
3.2.7 0.2% silver nitrate solution is reused 3-4 times, and the silver staining time is appropriately extended every time；
3.2.8 because not easy to clean, dress silver nitrate, developer solution cannot be mixed with the basin of fixer and fixing solution；
3.2.9 after electrophoresis, after smearing the glass plate of vaseline, lath hot-water soak, vaseline is wiped with paper, then clean
When be easier to clean, or impregnate after cleaned with dish washing liquid；
4 photograph
With gel image analyser photograph or dry glue scanning.
2. SSR amplified production native polyacrylamide gel electrophoresis method according to claim 1, which is characterized in that
(1) Acr and Bis is never poison, is irritating to the skin effect, and when operation should wear gloves and mask；(2) glass plate table
Smooth pieces are answered in face, otherwise be will cause in electrophoresis and are generated bubble between gel slab and glass plate；(3) sample template comb teeth should be put down
It is whole smooth；(4) there cannot be a bubble when filling gel, in case electric current passes through when influencing electrophoresis；(5) it is sure not to destroy sample-adding bottom portion of groove
It is smooth, in order to avoid electrophoresis back zone band distort；(6) lath must be consistent with comb thickness, and glue film otherwise will be generated in loading wells,
Influence point sample and last electrophoresis quality；(7) glue should be chalcogenide glass with glass plate；The height of the loading wells of (8) two hectographs
Difference only has 0.5cm, and when point sample, which should be noted that, to be avoided o'clock on the point to another hectograph of the sample mistake on a hectograph；
(9) due to simultaneously electrophoresis hectograph number it is more, Marker point on different swimming lanes, to distinguish different hectographs；(10)
Constant pressure or constant current electrophoresis, constant current；2 offset plates of electrophoresis, electric current are set as 100-120mA, voltage 300-400V；4 offset plates of electrophoresis,
Electric current is set as 200-240mA, voltage 300-400V；But voltage should be kept constant, otherwise band is easily bent.
3. a kind of device for realizing SSR amplified production native polyacrylamide gel electrophoresis method described in claim 1,
It is characterized in that, including 3 pieces of glass plate, adhesive tape 4, comb 1,2, clip；
Three pieces of glass plates outer dimension used is long and wide all identical, and rectangular glass is having a size of 20cm × 17cm；Scrobicula glass plate
To there is the notch of a recessed 1.5cm × 17cm in a long side；Deep concave glass plate has the notch of a recessed 2.0cm × 17cm,
The thickness of glass plate is all 3mm；Adhesive tape length is 18cm, broadband 1cm, the hectograph thickness adjustment that thickness makes as needed；
The thickness of comb is identical with the thickness of adhesive tape, the number of teeth have 15,20,40 it is not, selected according to requirement of experiment；
First rectangular glass is placed on cushion block when making hectograph, then puts one respectively close to marginal position in two short side
A adhesive tape, adhesive tape short side and does not have jagged glass plate bottom edge to put together, and the short side of long side glass plate is parallel, on two adhesive tape
Scrobicula glass plate is put, then similarly an adhesive tape is being put respectively close to scrobicula glass plate short side edge, then again depth
Concave glass plate is placed in adhesive tape；It is required that the notch of two pieces of recess glass plates is in the same direction, the other three of three pieces of glass plates
While stacking neatly, two interlayers are formed between three pieces of glass plates in this way, is finally clipped in respectively with two clips and is stacked in one
Two short side middle positions of the three pieces of glass plates risen；Under laid-flat status, the separation gel prepared is poured into two from recess side
In interlayer, plugs comb and put on the support frame after glue polymerization completely to be concentrated, clamp fixation with clip, lightly take out comb
Son cleans loading wells with distilled water immediately, just forms the hectograph with two pieces of separation gels；Other one piece is made in the same way
Hectograph is individually placed to the both sides of vertical slab electrophoresis groove, with clamp, toward the buffer chamber up and down of electrophoresis tank in that electrode is added is slow
Sample is clicked and entered sample well bottom with microsyringe, carries out electrophoresis by fliud flushing.
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| CN109307703A (en) * | 2017-07-26 | 2019-02-05 | 贵州大学 | The native polyacrylamide gel electrophoresis method of Rosa roxburghii Tratt SSR marker polymorphic detection |
| CN109609494A (en) * | 2018-12-26 | 2019-04-12 | 广西壮族自治区农业科学院 | A kind of separation method of the pcr amplification product based on SCoT label |
| CN114380943B (en) * | 2022-01-11 | 2023-04-21 | 北京志道生物科技有限公司 | Polyacrylamide gel and preparation method and application thereof |
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|---|---|---|---|---|
| CN1078975A (en) * | 1993-06-25 | 1993-12-01 | 南京农业大学 | Production technology of polyacrylamide gel for plate electrophoresis and glue pouring device |
| WO1997043630A1 (en) * | 1996-05-17 | 1997-11-20 | Purdue Research Foundation | Miniaturized disposable gels for dna analysis |
| JP2004510168A (en) * | 2000-09-29 | 2004-04-02 | プロテオム システムズ リミテッド | Electrophoresis system |
| CA2288123C (en) * | 1998-04-17 | 2004-07-06 | The Perkin-Elmer Corporation | Electrophoresis method and apparatus for separating bio-organic molecules |
| CN101614695A (en) * | 2009-07-23 | 2009-12-30 | 杭州吉来生物技术有限公司 | A kind of integral electrophoretic apparatus that is used for gel electrophoresis |
| CN202814913U (en) * | 2012-10-15 | 2013-03-20 | 刘建强 | Gel containing disposable electrophoresis apparatus with replaceable lanes |
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2014
- 2014-05-30 CN CN201410234799.0A patent/CN103995039B/en not_active Expired - Fee Related
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1078975A (en) * | 1993-06-25 | 1993-12-01 | 南京农业大学 | Production technology of polyacrylamide gel for plate electrophoresis and glue pouring device |
| WO1997043630A1 (en) * | 1996-05-17 | 1997-11-20 | Purdue Research Foundation | Miniaturized disposable gels for dna analysis |
| CA2288123C (en) * | 1998-04-17 | 2004-07-06 | The Perkin-Elmer Corporation | Electrophoresis method and apparatus for separating bio-organic molecules |
| JP2004510168A (en) * | 2000-09-29 | 2004-04-02 | プロテオム システムズ リミテッド | Electrophoresis system |
| CN101614695A (en) * | 2009-07-23 | 2009-12-30 | 杭州吉来生物技术有限公司 | A kind of integral electrophoretic apparatus that is used for gel electrophoresis |
| CN202814913U (en) * | 2012-10-15 | 2013-03-20 | 刘建强 | Gel containing disposable electrophoresis apparatus with replaceable lanes |
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