Chapter 2 DNA Mao slice and gel electricity Yong
The first upright conduct say
A. Slice Mao Mao to slice analysis inside DNA restriction
Limitting to slice Mao inside sex can especially foreign land’s combine be called restriction Mao to identify the DNA sequence of sequence at 1 inside or its neighborhood of especially the different order up, and incise a pair of chains DNA.It can is divided into 3 types: Ⅰ and Ⅲ Mao are in same protein member and have already incise with polish(A Ji turn) a function and depend on at the ATP existence.The Ⅰ Mao combine in identify a DNA that point combines to random incise to identify a point not distance, but the Ⅲ Mao incise DNA member in identifying a point, then the solution leave from the bottom thing.Ⅱ Constitutes to°from two kinds of Maos: In order to limit to slice nucleic acid Mao(restriction Mao) inside sex 1 kind, it incise some an especially different pit Mao sour sequence; Another turn Mao for the independent A Ji, it polish same identify a sequence.Sliced Mao to get in the member gram the Long inside the restriction in Ⅱ extensively applied, they are the foundations which reorganizes DNA.The great majority Ⅱ restriction Mao identifies length to check Mao sour reply for 4-6 symmetry especially different pit Mao sour sequence(such as the EcoR Ⅰ identify six sequences with sour pit Maos:5′- The G ↓ AATTC-3′), there are a handful of Maos identifying longer sequence or Jian3 Bing4’s sequence.The Ⅱ Mao incises a point in identify the sequence and have of incise in the symmetry stalk, produce the DNA part of even bitter end;(like Sma Ⅰ :5′-the CCC ↓ GGG-3′)Have of incise a point is one side at the symmetry stalk, the DNA part which produces to take to have single chain outstanding bitter end call gluing sex carried and produce after incise identify a sequence such as the EcoR Ⅰ 2 with each other repair of glue sex bitter end.
5′…G ↓ AATTC …3′ →5′… G AATTC …3′
3′…CTTAA ↑ G …5′ →3′… CTTAA G …5′
Slice Maos inside the DNA pure degree, buffer liquid, temperature condition and restriction will influence to slice the activity of Mao inside restriction.Slice Mao to be free from RNA or the influence of the single chain DNA inside greatly part of restrictions.When the little pollutant gets into restriction to slice Mao to store to save liquid China Times, will influence it to use further, so slice Mao in absorb the restriction, want to use a new straw head each time.If adopt to slice Mao inside two kinds of restrictions, necessarily claim attention to provide respectively the most suitable salinity respectively.If both can use same buffer liquid, then can water solves in the meantime.If the demand different salinity, then slicing Mao inside the restriction of low salinity has to use first, regulate salinity later on and use again to slice Mao water solution inside the restriction of high salinity.Can also use after the first Mao slice reaction completion etc. the physical volume Fen/chloroform take out to lift and add 0.100% physical volume 3 mols/L NaAc and 200% physical volumes have no water ether, mix to evenly postpose-70 ℃ low temperature refrigerator 30 minutes, leave heart, dry lay equal stress on lately is dissolve in a buffer liquid juniors to go the second Mao to slice reaction.
Slice Mao Mao to slice a diagram and call a DNA physical diagram inside the DNA restriction, it slices Mao Mao to slice a point to constitute inside various restrictions make sure by a series of position and mean by straight line or the wreath form diagram type.Slice inside the establishment restriction in the DNA sequence the analysis, the function diagram of gene set draw, the set up of DNA cloning, gene library etc. the work the Mao diagram is an all indispensable link, the development gets up in recent years of the RFLP(the restriction part length many Tais) technique is also an establishment at it of foundation up.
Set up to slice a Mao diagram to contain many methods inside the DNA restriction.Usually combine to use to slice Mao inside various restrictions, pass comprehensive analyze various Mao a list to slice and various Maos of different combination slice to gain in the meantime of the restriction part size to make sure various Mao of the Mao slice a point and it opposite position.The Mao slices the use value of diagram to depend on in it of accuracy and accurate degree.
In the Mao slice the diagram creation the process, for acquiring to take clear electricity Yong a diagram, the general DNA dosage is about 0.5-1 μ gs.The Mao solution which limits to slice Mao inside sex respond the most suitable condition is each not same, various Mao has the Mao that it correspond to slice a buffer liquid with the most suitable reaction temperature.(mostly is 37 ℃ )Confront a grain of DNA Mao slices reaction but speech and limit to slice Mao dosage inside sex can press the standard system 1 μ g the DNA add one unit Mao and digest for 1-2 hours.But want the dosage that the complete Mao solution has to then increase Mao, generally increase 2-300%, even more, respond that time also wants appropriate extension.
Two. The gel gives or get an electric shock Yong
The Qiong fat sugar or the polypropylene Mao An gel electricity Yong are purely the standard methods that the separation authenticates and turns DNA part.That technique operation is simple and fast, can distinguish to use the DNA part that other methods(such as density steps the degree leave a heart method) can’t separate.When imbed dyestuff bromine to turn B Ding with the fluorescence of low density(Ethidium bromide, the E dye and the DNA that can check a 1-10 ngs at least under the purple outside light take, can make sure that the DNA part is in the position in the gel thus.In addition can recover particular DNA to take still from the gel in electricity Yong, used for a later gram a Long operation.
The Qiong fat sugar and polypropylene Mao An can make into various shape, size and hole degree.The Qiong fat sugar gel separates the DNA slice degree all of the scopes more wide, the different density Qiong fat sugar gel can separate length from 200 bps to the DNA part of near 50 kbs.The Qiong fat sugar usually uses level device in strength with direction Heng settle of the electric field descend electricity Yong.The polypropylene Mao An separates crumb segment’s DNA(5-500 bps) effect better, it explains dint very high, even differing the DNA part with 1 bp can separate.The polypropylene Mao An gel electricity Yong is very quick, can accept a great deal of opposite DNA, but making and operation compare the Qiong fat sugar gel difficult.The polypropylene Mao An gel adopts perpendicular device to carry on giving or get an electric shock Yong.Currently, the general laboratory uses the flat panel gel of the Qiong fat sugar level electricity swimwear more to place to carry on a DNA electricity Yong.
The Qiong fat sugar is mainly a kind of solid in the DNA the making the electricity Yong support Ji quality, its density is decided by a Qiong fat the density of the sugar.In the electric field, just under the neutral pH value take the DNA of negative charge a facing the sun pole migration, its migration velocity from following and various factor decision:
1, DNA member size:
Line form double chain DNA the member becomes inverse ratio in the certain migration velocity within gel of the density Qiong fat sugar and the DNA molecular weight logarithms, the member is more big then the dint get difficulty be more big, also more difficult go in the gel hole at the Ru, as a result move more slow.
2, the Qiong fat sugar density
A line form DNA member which gives to settle size, its migration speed is each not same in the Qiong fat sugar gel of different density.Logarithms and gel density of the DNA electricity Yong migration rate become line sexual behavior.The choice of gel density is decided by the size of DNA member.Separate the gum density that less than 0.5 kbs DNA parts need is 1.2-1.5%, separate the gum density that more than 10 kbs DNA members need is 0.3-0.7%, the DNA part all ofs the gum density need then is 0.8-1.0% between both.
3, the Gou elephant of DNA member
When the DNA member is placed in different Gou elephant, it is in the electric field move distance not only relate to molecular weight, return with its Gou elephant relevant.The line form of same molecular weight, open wreath with super and spiral DNA to move speed in the Qiong fat sugar gel is different, the super and spiral DNA moves most quickly, but line form double the chain DNA move most slowly.Such as there are several DNAs take in giving or get an electric shock Yong to authenticate a quality grain a pure degree discovering gel hard assurance is quality grain the Gou elephant with different DNA cause still because imply other DNAs to cause, can take to pursue a recovery from the Qiong fat sugar gel full general DNA, use to slice a Mao difference water a solution inside the same kind of restriction, then give or get an electric shock Yong, if appear a same DNA diagram on the gel, then is the same kind of DNA.
4, power supply electric voltage
At low electric voltage, the migration velocity of the line form DNA part and the electric voltage add becomes a direct proportion.But along with the increment of the electric field strength, the migration rate of the DNA part of different molecular weight will be increase with different range, the part is more big, strong because of field go up a causable migration rate to go up range also more big, so electric voltage increment, the Qiong fat sugar gel of valid separate the scope will contract.Want to make the resolution of more than 2 kbs DNA parts attain biggest, the electric voltage add can not be over the 5 vs/cm.
5, imbed the existence of dyestuff
The fluorescence dyestuff bromine turns B Ding to used for examine the Qiong fat sugar gel in of DNA, the dyestuff will imbed to pile up of alkali Ji to its and make longer a line form and take the wreath form of indentation DNA, make it rigid and stronger, will also make the line form DNA migration the rate lower 15%.
6, the ion strength influence
Give or get an electric shock the Yong buffer constitute of liquid and the DNA the electricity Yong migration rate of the its ion strength influence.While have no ion existence(such as misapplication the distilled water prepare gel), electric conductivity rate minimum, the DNA scarcely moves, in the buffer liquid of high ion strength(if the mistake adds a 10 ×s electricity Yong buffer liquid), the electric conductivity then is very high to also obviously produce hot, the serious hour will cause gel to melt or the DNA change sex.
For natural double chains DNA, a few in common use electricity Yong buffer liquids contain TAE[contain EDTA(pH 8.0) with Tris-acetic acid], TBE(Tris-boric acid and EDTA), TPE(Tris-phosphoric acid and EDTA), generally prepare concentrated female liquid, keep at the indoor temperature.
Section 2 material, equipments and try
A, material
λ DNA: Purchase or withdraw by oneself to purely turn; Reorganize the pBS material or pUC 19; EcoRI Mao and its Mao slice a buffer liquid: Purchase finished product; The Hind Ⅲ Mao and its Mao slice a buffer liquid: Purchase finished product;Qiong fat sugar(Agarose): The importing or domestic electricity Yong uses a Qiong fat sugar all can.
Two, equipments
The level type electricity swimwear place, electricity Yong instrument, set type high speed leave scheming, constant temperature water bath pot, little by little move liquid gun, microwave oven or electric stove, purple outside deeply shoot an instrument, take a picture support, camera and its enclosure.
Three, try
1 and 5 × TBEs give or get an electric shock a Yong buffer a liquid:The formula sees chapter 1.
2 and 6 × electricity the Yong carry a kind buffer a liquid:0.25% bromine powders are blue, 40%(w/v) cane sugar aqueous solution, store to save at 4 ℃ .
3, the bromine turn B tablet(the E aqua female liquid:prepare EB 10 mgs/ml, use aluminum screen or black paper package container, keep at the indoor temperature then.
Section 3 operates a step
A, the DNA Mao slice reaction
1, will sweep dry combine through eppendorf tube(had better 0.5 mls) of put out the germ serial number, use to little by little move the liquid gun joins DNA one μ g respectively with correspond of inside restriction slice Mao to respond 10 × buffer the liquid be 2 μ ls, again join heavy steam water to make total accumulate to 19 μ ls, will tube inside aqua after mix evenly join one μ l Mao liquid, by hand point to flip tube wall to make the aqua mix evenly, can also use to little by little leave scheming to jilt once, make aqua the concentration is take care of bottom.This operation is the whole key which test success or failure, prevent°from wrong add, leak to add.Should as far as possible reduce time that it leaves refrigerator while using to slice Mao inside restriction, the in order to prevent activity lower.
2, place an eppendorf tube on the appropriate support thing(if put plank at the foam plastics up) after mixing to evenly respond system, 37 ℃ water bath heat preservation 2-3 hour, make the Mao slice reaction completely.
3, take care of to join each time 2 the μ l 0.1 mols/L EDTA(pH 8.0), mix evenly to stop respond, place at the conservancy in the refrigerator to provide for use.
Two, the make of standard of the DNA molecular weight
Adopt EcoR Ⅰ or the Hind Ⅲ Mao solution gain of the part of λ DNA come Be give or get an electric shock Yong of molecular weight standard.The λ DNA is a length the chain DNA is about the double of the 50 kbs member, the its merchandise aqua density is a 0.5 mgs/ml, Mao solution reaction the operation as above says.The HindIII gets 8 parts after incise the DNA, the length distinguishes to 23.1, 9.4, 6.6, 4.4, 2.3, 2.0, 0.56 with 0.12 kbs.The EcoRI gets 6 parts after incise the lDNA, the length distinguishes to 21.2, 7.4, 5.8, 5.6, 4.9 with 2.5 kbs.
Three, the Qiong fat sugar gel make
1, take 5 × TBE buffer liquid 20 mlses to fill with water to the 200 mls, prepare 0.5 the × TBE dilution buffer liquid, need to be use.
2, the making of gum liquid:Call to take the 0.4 g Qiong fat sugar, place in the 200 ml taper bottle, join a 50 mls 0.5 the × TBE dilution buffer liquid, put to heat to Qiong fat all of the sugars to melt into the inside(or electric stove up) of the microwave oven, take out to shake evenly, this is 0.8% Qiong fat sugar gel liquid.Heat in the process want to wave often, make to attach to get into aqua at bottle the Qiong fat sugar grain of the wall.Should cover a film while heat, evaporate by decrease water.
3, the gum knothole make:Use organic glass gum slot both ends the rubber cream(the breadth is about the 1 cm) is close to seal respectively.Will seal good gum slot to place to support a thing at the level up, put on a sample comb, the attention observation comb Chi descends good luck and should keep the 1 mm or so cleft with the gum slot bottom noodles.
To cool off to 50-60 ℃ Qiong fat sugar gums join bromine to turn B tablet in the liquid(the E aqua make it eventually a density for 0.5 μ g/mls.(can also not join the EB into the gel, but give or get an electric shock the Yong uses 0.5 μ g/mls again behind of the EB aqua soak dyeing)Use to move a liquid machine to absorb the jot melt of the Qiong fat sugar gel seal rubber cream seamy side, after needing the Qiong fat sugar aqua to solidify the Qiong fat of the surplus the sugar carefully pours into gum slot and make the gum liquid become an even gum layer.Pour gum of the temperature can’t be too low, otherwise solidify asymmetry, speed also can’t too quick, otherwise easily appear a spirit bubble.Stir a comb after needing gum to completely solidify, notice don’t hurt gel of comb the bottom, then to slot inside join 0.5 the × TBE dilution buffer the liquid by luck didn’t lead gum plank a top surface to the liquid noodles.Because the edge effect sample slot neighborhood will have some swell up, obstruct a buffer liquid into sample slot, so want to notice assurance sample slot in should fill with a buffer liquid.
4, add kind:Take 10 the μ l Mao solution liquid with 2 μ l 6 ×s carry a kind liquid to mix evenly and use to little by little move a liquid gun to carefully join sample slot.If the DNA content be low, then can increase the last amount of kind according to above-mentioned comparison, but total accumulate can’t exceed sample slot capacity.Finish add a sample and replace a tip head each time, in order to prevent mutual pollution, want to carefully operate while noticing the last kind, avoid damage gel or pierce the sample slot bottom gel.
5, give or get an electric shock Yong:Close electricity Yong slot a cover after finishing add kind, immediately connect power supply.Control the electric voltage keeps in the 60-80 Vs and the electric current is above in the 40 mAs.When the bromine Fen blue took to move to follow about the 2 cms before being apart from gel, stop electricity Yong.
6, dyeing:Didn’t add EB gum plank to move to go into the EB aqua of 0.5 μ g/mls after giving or get an electric shock Yong to complete, under the indoor temperature dye for 20-25 minutes.
7, observation with take photo:After the wave-length is the long wave with 254 nms grows purple outside in the light to observe to dye of or have already added to have EB of the electricity Yong gum plank.The DNA existence displays naked eye can the Jie red fluorescence of Bian take.The purple light should put on protection glasses or organic glass mask when the purple light by lamplight observes, the in order to prevent hurt eyes. After camera lens plus nearly shooting mirror slice and red color filter is fixed camera in take a picture up, adoption pan film, diaphragm 5.6, expose time 10-120.(according to fluorescence the depth choice for take)
8, DNA molecular weight standard the creation of the curve:Take sample slot as point of departure in the electricity Yong photograph enlarge, slice with EcoR Ⅰ and Hind Ⅲ Mao that the card Chinese foot measures λ DNA part of move distance, take Li rice as unit.Take checking Mao the common logarithm that is sour number as Zong to sit a mark, take move distance as horizontal sit a mark, draw the at all point smooth curve of a link in sitting mark paper, is should give or get an electric shock DNA molecular weight under the Yong condition of standard curve.
9, the DNA Mao slice segment the measurement of the size:Mete out DNA sample with the card Chinese foot in the electricity Yong photograph enlarge each part of move distance, according to this number, look up a homologous logarithms value on the standard curve of the DNA molecular weight, compute further an each part of molecular weight size.(if sit mark paper to draw standard curve with the single logarithms, then can according to move distance to directly look up the size of DNA part)Whereas, if have already known the size of DNA part may also is up looked up it to anticipate by the standard curve of move distance.
10, the assurance which line up row order of the DNA Mao slice segment:Slice according to the single Mao, the double Mao slices the electricity Yong which slices with many Maos analytical result, the data which checks against the DNA Mao slice segment size carries on logic to reason logically, then makes sure that each Mao slices the opposite position that scrappy row row order and each Mao slice a point.Mean by the wreath form diagram or line graph, then become that DNA member of slice a Mao diagram inside restriction.
[Notice]1.The DNA aqua physical volume that Mao add when slice can’t be too big, otherwise other compositions in the DNA aqua would the interference Mao respond.
2, Mao vitality usually means with the Mao unit(U), the definition of Mao unit BE:At the most suitable reaction under the condition, completely decline to solve the 1 mg lDNA Mao quantity as an unit for an hour, but many experiment make of DNA not the elephant lDNA is easy to decline a solution so and need appropriate increment Mao of usage quantity.The Mao which responds to join a surfeit in the liquid isn’t suitable, in addition to consider cost, little by little miscellaneous quality possible interference subsequent reaction in the Mao liquid.
3, market sale of the Mao general density very big, rise to see for the economy, usage can in advance carry on dilution with the Mao reaction buffer liquid(one ×).Moreover, the Mao usually keeps in 50% glycerin, in the experiment, in response to reaction the glycerin density in liquid control at 1/10 under, otherwise, the Mao activity will be influence.
4, observation the DNA can not get away from purple outside deeply shoot an instrument, but the purple outside light have already incised a function to the DNA member.From glue together recovery DNA, should as far as possible shorten light shine on time combine the adoption long wave grow a purple and outside light(300-360 nms) to incise by reducing purple and outside light DNA.
5, the EB be strong to guide to change also medium etc. toxicity, prepare with usage hour should wear gloves, and don’t spread the EB to table’s top or ground.Every be stained with dirty EB container or product have to after specialized processing then can clean or throw away.
6, be EB too many, the gum once dyed deeply, when the DNA took to see not pure, can put the gum into distilled water to pour, observe again after 30 minutes.
Think subject of examination
1. Do you think and may be the reason why if a DNA Mao solution liquid discovers after give or get an electric shock Yong that the DNA isn’t slice to move?
2. The DNA member migration rate is subjected to in the Qiong fat sugar gel electricity Yong which factors of influence?
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