分子细胞遗传学常用实验技术精.docx

分子细胞遗传学常用实验技术精.docx

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分子细胞遗传学常用实验技术精

分子细胞遗传学常用实验技术

GeneralLaboratoryGuidelines

A.Beforestarted:

Beforeyouenterthelaboratory,makesureyouhavereadandfullyunderstandthelaboratorysafetymanual.Pleasereadthelaboratorymanualbelowandfollowtheguidelinesofeachexperiment.KeepinmindthatalltheguidelinesinthismanualareINGENERALbutnotforeverydetailsteps.Makesureyouhavefullyunderstoodeachstepintheprotocolanditsreason.Pleaseaskyoursupervisorifyouhaveanythingunclear.

B.Solutions-Ifyouuseasolutionandthevolumeislow（lessthan500ml）,notifythepersonwhomakesit.Donotwaituntilthereisalmostnoneleft.Ifyouanticipateusingalotofaspecificsolution,youmaywanttomakesomeforyourself.Makesureallsolutionsarelabeledwithyourinitials,dateandthechemicalnameandconcentration.

C.Pipettipsandmicrofugetubes-Afteryouuseaboxofpipettips,refillitandplaceitontheshelf.Whenseveralaccumulatetechnicianwillautoclavethemandreturntothedrawer.Keepyourplasticboxofmicrofugetubes.Youcanjustrefillyourboxasnecessary.Don’taccumulatemanypipetboxeswithoutrefillingthemorotherswon’thaveanytouse.

D.Autoclave-Donotautoclavewasteitemswithsterileitems.

E.Orders-Ifyouseethatsomethingisrunningloworifyouanticipateusingalotofanitem,notifytheresponsiblepersonandhewillplacetheorder.Donotwaituntiltheitemisgoneoralmostgone-givetheorderinadvance.

F.LaminarFlowHood-Makesureyousprayitdownwith70%ethanolbeforeandafteryouuseit.Ifthehazardouswasteisfilled-autoclaveit.Donotletitoverfill-thewasteshouldbedisposedofweekly.

G.Dishes-Wehaveaverysmallsinksowashyourglasswareeveryday.Washwithsoapandwater.Rinsewithdistilledwater.Whenyouritemshavedried,putthemaway.Don’tletitemsaccumulatenearthesink.Thesamegoesforyourbench-cleanupaftereveryuse.

H.Equipment-Ifyouhaveaproblemwithanyequipment,notifytheresponsiblepersonimmediately.Beforeusinganyequipment,askfordirectionsorreadthemanualfirst.

I.Pipets-Donotsetpipetsovertheirspecifiedrange.Pipettingsolutionupanddownslowly.Takegreatcarewhenpipetschemicalslikechloroform.Refertothefactorywebsiteforinstructionsonhowtofixbrokenpipetsortheinstructionbooklet.

P-20:

2-20ulP-200:

50-200ulP-1000:

100-1000ul

J.RefrigeratorandFreezer-Alwaysmakesurethedoorstotherefrigeratorsandfreezersareclosedwhenyouarefinishedwiththem.

K.NylonMembranes-AllSouthernfiltersshouldbewrappedinsaranwrapandstoredinasealedziplockbagtopreventmoistureloss.Makesurethebagislabeled“Stripped”iftheyhavebeenusedbeforeandarereadyforre-probing.

L.Opensampleorregents’tubes:

Alwaysdoashortspinbeforeyouopenanysampleandregenttubestoeliminateanypossiblecontamination.

M.TransferhighconcentrationDNAsamples:

DuringDNAextractionandafterwardpurification,makesureyourcutofftheendofthepipettips.ThesharpendoftipswillbreaktheDNAmolecular.

第一部分常用试剂、培养基的配制

0.5MEDTApH8.0

EDTA2Na2H2O186.1g

NaOH～20g

H2O→1L

3MNaAcpH5.2高压灭菌

NaAc3H2O408.3g

醋酸调节pH至5.2

ddH2O800ml

1000ml

5MNaCl

NaCl146.1g

ddH2O→500ml

10%SDSpH7.2

SDS20g

HClafewdrops

200ml

1MTris-HClpH8.0

Trisbase121.18g

HCl～42ml

ddH2O→1000ml

TEpH8.0

Tris-HCl（pH8.0）10ml

EDTA2ml

ddH2O→1000ml

TE0.1pH8.0

Tris-HCl（pH8.0）10ml

EDTA200μl

ddH2O→1000ml

50×TAE

Trisbase242g

冰醋酸57.1ml

0.5MEDTA（pH8.0）100ml

ddH2O→1000ml

10×TBE

Trisbase108g

硼酸55g

0.5MEDTA40ml

ddH2O→1000ml

1MKCl（M.W.74.5）

KCl7.45g

ddH2O→100ml

Gel-loadingBuffers（foragarosegel）

溴酚蓝（0.25%）100mg

蔗糖20g

ddH2O→50ml

E.B.10mg/ml

E.B.1g

ddH2O100ml

HCl（1N11.6M/L）

36.5%HCl86.2ml

H2O913.8ml

1M葡萄糖（M.W.180）

葡萄糖18g

ddH2O→100ml

过滤除菌，0.22μm

1MMgCl2（M.W.203.3）注意：

吸湿性强，空气中迅速潮解，请迅速配制

MgCl26H2O20.32g

ddH2O→100ml

灭菌

1MMgSO4（M.W.246.5）

MgSO424.64g

ddH2O→100ml

不易溶解，需要使用搅拌器

灭菌

IPTG储存液（M.W.238.3）

2g溶解于8mlddH2O中，通过0.22μm过滤器，过滤除菌，调整至10ml。

分装，保存于-20℃。

X-gal储存液

20mg/ml二甲基甲酰胺于玻璃瓶或聚丙烯管中，用铝箔包裹严密，不需要过滤除菌，

保存在-20℃。

抗生素

抗生素

浓度

保存条件

氨苄青霉素

50mg/ml（溶于水）

-20℃

卡那霉素

10mg/ml（溶于水）

-20℃

氯霉素

50mg/ml（溶于乙醇）

-20℃

以水为溶剂的抗生素储存液应用0.22μm过滤器，过滤除菌

用乙醇溶解的抗生素溶液无需除菌处理，所有的抗生素贮存液都应放于不透光的容器中保存

20×SSC

NaCl3M（58.44×3=175.32）

柠檬酸纳0.3M（294.1×0.3=88.23）

用1NHCl调节pH（7.0）

10×PBS

NaCl1.3M（58.44×1.3=75.97）

Na2HPO40.07M（141.96×0.07=9.94）

NaH2PO40.03M（120×0.03=3.6）

（NaH2PO4H2O137.99×0.03=4.14）

Na2HPO42H2O0.07M（358.14×0.07=25.07g）

NaH2PO42H2O0.03M（156.01×0.03=4.68g）

柠檬酸buffer0.01MpH4.5

Na3C6H5O72H2O1.47g

C6H8O71.05g

ddH2O→500ml

鲑鱼精DNA制备（salmonspermDNA）

1Dissolve1gDNA（anyfishspermDNA）in100ml0.4MNaOHovernight

2Boil30mins（片断长度为200-1000bp）

3Chillneutulizewithconcentratedglacialaceticacid

（～2ml）

4Spinatdehriifany

5Add2VEtOHincubate-20℃for≥1h

6PelletDNA-washwith70%EtOHdry

7Resuspendin100mlTE=10mg/ml

SephadexG-50

AddSephadexG-50（medium）todistilledsterilewater.WashtheswollenresinwithH2Oseveraltimestoremovesolubledextran,whichcancreateproblemsbyprecipitatingduringethanolprecipitation.Finally,equilibratetheresininTE（pH7.6）,autoclave（101b/sqinfor15min）andstoreatroomtemperature.

培养基的配制

SOC

SOB10ml

Glucose（1M）200μl

MgSO4（1M）100μl

MgCl2（1M）100μl

SOB

Bactotryphone2g

Yeastextract0.5g

5MNaCl0.2ml

1MKCl0.25ml

5NNaOH～0.02ml

ddH2O→100ml

高压灭菌

LB培养基

tryphone10g

Yeastextract5g

NaCl10g

ddH2O→1000ml

5NNaOH～0.2ml

固体LB培养基1L

向液体培养基中加入1.5%（1000ml→15g）的琼脂,并且加入：

X-gal（2%）1000μl

IPTG（20%）100μl

2×YT

tryphone16g

Yeastextract10g

NaCl5g

如果需要用1NNaOH（～1ml）调节pH至7.0

ddH2O→1000ml

第二部分常用技术

植物基因组DNA的提取

1取0.1-0.2g植物新鲜幼嫩的组织于2mlEppendorf管中，浸没在液氮里并用竹棍研磨。

2向管加入3颗钢珠并且剧烈旋涡振荡直至叶片成粉末状。

3将钢珠取出，加入700μl预热的提取buffer，彻底混匀，65℃温浴10min。

（每3min温和混匀一次）

4加入等体积1:

1的酚/氯仿（每试剂350μl）彻底混匀（至少2min），离心13200rpm,5min。

5将上清转移至一干净的管子中，并加入等体积的氯仿（700μl）和2μlRNase（10mg/ml）,彻底混匀5min。

离心13200rpm,5min。

6将上清转移至一干净的管子中，并加入0.7体积的异丙醇，温和但彻底混匀直至DNA纤维出现。

7将DNA纤维勾出，并用70%的酒精洗涤DNA两次。

8在室温下将DNA晾干，并用适当体积的TE溶解。

附录：

DNA提取Buffer

1MTris-HCl（pH7.5）10.00ml

5MNaCl2.50ml

0.5MEDTA2.50ml

20%SDS2.50ml

2-巯基乙醇150μl（用时再加）

ddH2O→50ml

高压灭菌

DNA纯化（酚：

氯仿：

异戊醇）

GenomicDNACleanupusingPCI

Preparation:

IftheDNApelletisinpurifyingbuffer,pouroffthepurifyingbufferandrinsethepelletinicecold70%ethanolfor1minute.Centrifugetubesat12,000RPMfor5minutes.Pouroffethanolanddrythepellet.Resuspendin30-200lofTE（DependingonquantityofDNA）.ForPCIcleanup,theDNAshouldnotbehighlyconcentratedortherewillbeagreaterlossofDNA.Treatsampleswith5lofRNAse（10mg/ml）per100µlofDNA.Placein37oCwaterbathfor30minutes.Tostopthereactionplacein65oCwaterbathfor15-20minutes.

Remembertoidentifyeachsampleclearlyonthesideandtopofeachtube.Makesurelabelisnotremovedduringeachstepofthisprocedure.

1.AddanequalvolumeofPCI（phenol:

chloroform:

isoamylalcohol,25:

24:

1）totheDNAinTE.（Onlyuseequilibratedphenol,pH=8.0）*Note-thePCImixistoppedwithalayerofTE-donotpipetfromthisTElayeranddonotmixthe2layers.

2.Invertthemixtureatleast20timessothatthecomponentsarethoroughlymixed.Itissometimesnecessarytoinvertvigorously（notrecommended）-makesurethecomponentsarenolongerseparatedinto2layersbeforespinning.

3.Spintubesat10,000RPMfor10-12minutes.Duringspin,labelasecondsetoftubes.

4.Cuttheendofthetips.Immediatelyafterspinning,slowlypipettheDNA（foundintheupper,aqueouslayer）andplaceinthesecondsetofclean,labeledtubes.ItisimportanttonotdisturbtheinterphasewhileremovingtheDNA.

5.Addanequalvolumeofchloroform:

isoamylalcoholtotheDNAjustremoved.

6.Invertthemixtureasbeforeandspinagainat10,000RPMfor10-12minutes.Labelafinalsetofcleantubes.

7.Immediatelyafterspinning,usingcuttipstopipettheDNA（foundintheupper,aqueouslayer）intotheclean,labeledtubes.

8.Add2volumesofcold95%ethanoland1/10volumeof3MNaoAc,pH5.2.Inverttubestomixcomponents.

9.Placetubesin–80oCfor30minutes.

10.Spintubesat12,000RPMfor12minutes.Decantthesupernatantanddrainthoroughly.

11.WashtheDNApelletwith1000loficecold70%ethanolfor1minutetoremoveresidualsalts.Centrifugeagainat12,000RPMfor5minutes.Inverttubesandallowpellettodryonbenchfor3-4hoursorinhoodfor15minutesoruntilthereisnoremainingtraceofethanol.

12.Resuspendpelletin30-200lTE（dependingonquantityofDNA）.

Notesonchoosingwhichalcoholandsalttouseinprecipitation:

1.BothethanolandisopropanolcanbeusedtoprecipitateDNAwhenaddedwithmonovalentcations.Choiceofalcoholismostlydependentonthevolumeoftheaqueousphase:

use2volumesofethanolor1volumeofisopropanol.

2.EithersodiumacetateorammoniumacetatecanbeaddedtotheDNApriortoprecipitation.Sodiumchloridecanalsobeused.Thechoiceisdependentonsubsequentapplications:

residualsodiumionscaninhibitDNAligaseandammoniumcaninhibitbacteriophageT4polynucleotidekinase.MostofthesaltshouldberemovedbywashingtheDNAin70%ethanol.Thefinalconcentrationofsodiumacetateshouldbearound300mM.Finalconcentrationofammoniumacetate（pH7.0-7.4）shouldbe2-2.5M.

DNAQuantificationbyAgaroseGelElectrophoresis

1.Preparea0.8%agaroseminigelcontainingethidiumbromide.

2.Dilute