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