Labprotocols分子生物学实验方法技术汇总.docx
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Labprotocols分子生物学实验方法技术汇总.docx
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Labprotocols分子生物学实验方法技术汇总
LABORATORYPROTOCOLS
InstituteofCellBiologyandGenetics
[1]GeneralPCRProtocol
HereinthisLab,wedo25µlPCRreactionsin0.25mlmicrofugetubes.YoucandoPCRindifferentsizereactionvolumesandinsmallertubesaslongastheyfitinthethermocycler.
PCRisverysensitivetocontaminationfromoutsideDNAs.Stepsshouldbetakentoreducethechanceforcontamination,suchaswearinggloves,andnotspittinginthetubes.Assembleyourreactionsonice.
实验前准备:
PCR开始前,将SDW,10×Buffer,10mMdNTPs,10µMprimers,模板,MgCl2取出放在冰上。
依次加入上述反应物,加完后,再加酶(酶要放在冰上)。
酶用完后马上放回-20°C保存。
1.Gentlyvortex(涡流)andbrieflycentrifugeallsolutionsafterthawing(融化,解冻).
2.Add,inathin-walledPCRtube,onice.(按体积从大到小依次加入)
SDWto25µl
Buffer(10X):
2.5µl
dNTPs(10mM):
1µl
primers(10µM):
1µl
template:
Xµl
MgCl2(25mM):
1.5µl
Taqpolymerase(keepat-20°C)0.25µl
3.Gentlyvortexthesampleandbrieflycentrifugetocollectalldropsfromwallsoftube.
4.PlacesamplesinathermocyclerandstartPCR.
GeneralPCRcycylesareasfollows:
1cycle-4mins-94°C;
35cycles-30sec-94°C;
30sec-X°C*;
Xsec-72°C;
1cycle-10mins-72°C.
[2]PCRproductpurificationprotocol
(TaKaRaAgaroseGelDNAPurificationKitVer.2.0)
1.ExcisetheDNAfragmentfromtheagarosegelwithaclean,sharpscalpel.Cutthegelintosmallpieces.
2.Weighthegelsliceinacolorlesstube.Add3-5volumes(1%agarose,3volumes;1-1.5%,4;1.5-2%,5)ofDR-IBufferto1volumeofgel(100mg~100μl).
3.Mixthoroughly.Incubateat75°C
for6-10min(oruntilthegelslicehascompletelydissolved).Tohelpdissolvegel,mixbyvortexingthetubeevery2–3minduringtheincubation.
4.Afterthegelslicehasdissolvedcompletely,add0.5DR-IBuffervolumeofDR-IItothesampleandmix.IfDNAfragments<400bp,addisopropanol(异丙醇)toafinalvolumeof20%.
5.Placeaspincolumninacollectiontube.
6.TobindDNA,applythesamplefrom3tothecolumn,andcentrifugefor1minat12000g.Discardflow-throughandplacethecolumnbackinthesamecollectiontube.
7.Towash,add0.5mlofRinse(冲洗液)Atothecolumnandcentrifuge
for0.5min.Discardtheflow-through.
8.Add0.7mlofRinseBtothecolumnandcentrifugefor0.5min.Discardtheflow-through.
9.Gotostep7.
10.Placethecolumnintoaclean1.5mlmicrocentrifugetube.
11.ToeluteDNA,add25μlofBufferEBorH2Otothecenterofthemembrane.Letthecolumnstandfor1minandcentrifugefor1min.(Using60oCEBorH2OwillincreasetheDNAconcentration).
[3]RNAIsolationUsingTRIzol
*Using1mlTRIzolpersampleforNorthernblot,and0.5mlTRIzolforRT.
*DEPCwater(dissolveDEPC(200µl)in400mlwater,incubateitat37℃withshakingovernight,andsterilize灭菌).
1.Homogenization均匀化ofthesampleisbymortar研钵andpestle杵(pre-cooledwithliquidnitrogen).Transferthesampletothepestleandgrinduntilalayerofveryfinedustisallthatisleft(keepthesamplefrozen).Useaspatula刮铲(pre-cooledwithliquidnitrogen)totransferthedusttotheTRIZOLsolutionpreparedina1.5mltube.Vortexmixturethoroughly.(using50-100mgoftissueper1mlofTRIzol).
2.Centrifugesamplesinaswingrotorat12,000xgfor10minat2-8℃toremovemembranes,polysaccharides(多糖)andhighmolecularweightDNA.
3.Transfersupernatanttoanewtubeandincubatethesamplesfor5minatroomtemperature(RT)(15to30℃)topermitcompletedissolutionofnucleoproteincomplexes.
4.Add0.2mlchloroform(氯仿)per1mlTRIzol.Captubessecurelyandshakevigorouslybyhandfor15sec.IncubateatRTfor2-3min.
5.Centrifugesamplesinaswingrotoratamaxof12,000xgfor15minat2-8℃.Transfer~75-85%oftheupperphasetoafreshtubebutsavetheorganicphaseforDNA/proteinisolation.
6.PrecipitatetheRNAfromtheaqueous(水的)phasebyadding0.5mlisopropylalcohol异丙醇per1mlTRIzolusedforhomogenization.IncubatesamplesatRTfor10minandcentrifugesamplesatamaxof12,000xgfor10minat2-8℃.
7.Discardsupernatant(上清液)andwashpelletwith1ml75%ethanol(乙醇)per1mlTRIzolusedinhomogenization.Vortexandcentrifugesamplesatamaxof7,500xgfor5minat2-8℃.
8.AirdrytheRNApelletfor~5-10minbutdonotletitdrycompletelytoensuremaximumsolubility.DissolvetheRNApelletinDEPCH2O,andincubateitat4℃overnight.(Orat55-60℃for10min).
9.RNAQuantitation.Warmupspec.Add2μlRNAsampleto398μlautoclaved(高压消毒的)dH2O.Scansamplesfrom220-320nm.RNA(mg/ml)=A260×0.04×dilutionfactor.
10.Purity.A260/A280=1.8-2.1inTris.Inwateraratio>1.65isOK.
11.StoretheRNAsamplesat-20℃.(RNAcanbestoredat4℃overnightorsnap-freeze(快速冷冻)samplesinliquidNandstoreat-80℃forlong-termstorage).
[4]PromegaRTprotocol
器材和药品:
无RNase的灭菌的Eppendorf管,1μg的RNA,0.5μl的寡聚引物,无RNase的SDW,2.5μl的5×反应液,0.625μl的dNTPs,0.5μl的RNase抑制剂,0.5μl逆转录酶。
做实验之前,先将PCR仪设置成:
①70°C,5分钟;②42°C,60分钟;③70°C,15分钟。
1.InasterileRNase-freemicrocentrifugetube,add1ng–1μgtotalRNA,0.5μlOligo(dT)(10μM),Nuclease-FreeWatertofinalvolumeof8.375μl.
2.Heatthetubeto70°Cfor5minutes,thencoolquicklyonicefor5minutes.
3.Addthefollowingcomponentstotheannealedprimer/templateintheordershown:
5×ReactionBuffer(with50mMDTT)2.5μl
dNTPs,10mM0.625μl
Ribonucleaseinhibitor0.5μl
M-MLVRT(逆转录酶)0.5μl(50–100units)
4.Mixgently.Incubatethereactionat42°Cfor60minutes.
5.Optionally,inactivatethereactionbyheatingfor15minutesat70°C.ThecDNAcannowbeusedasatemplateforamplificationbyPCR.
[5]3’RACEprotocol(FirstChoice®RLM-RACE)
注:
3’RACE和5’RACE可根据购买的试剂盒说明书操作具体步骤
A.ReverseTranscription
<5ul
1 μgtotalRNAor50 ngpoly(A)RNA
2ul
dNTPMix
1ul
3' RACEAdapter
1ul
10×RTBuffer
0.5ul
RNaseInhibitor
0.5ul
M-MLVReverseTranscriptase
to10ul
Nuclease-freeWater
1.AssemblethefollowinginanRNase-freemicrofugetubeonice:
2.Mixgently,spinbriefly.
3.Incubateat42°Cforonehour.
4.Storereactionat–20°CorproceedtothePCRstep.
B.PCRfor3' RLM-RACE
The3' RACEOuterPrimerworkswellinPCRusinganannealingtemperaturefrom55to65°C.Therefore,anannealingtemperatureof60°Cisprobablyareasonablestartingpoint.Theoptimaltemperatureforyourprimerandtemplatecombinationmayhavetobedeterminedempirically.
[6]5’RACEprotocol(FirstChoice®RLM-RACE)
1.CalfIntestineAlkalinePhosphatase(CIP,小牛肠碱性磷酸酶)treatment
a.AssemblethefollowingcomponentsinanRNase-freemicrocentrifugetube:
Amount(ul)Component
Xul5 μgtotalor125 ngpoly(A)RNA
1ul10×CIPbuffer
1ulCalfIntestineAlkalinePhosphatase
To10ulNuclease-freeWater
b.Mixgently,spinbriefly.Incubateat37°Cforonehour.
2.TerminateCIPreactionandphenol:
chloroformextract
a.Add
Amount(ul)Component
7.5ulAmmoniumAcetate(醋酸铵)Solution
57.5ulNuclease-freeWater
75ulacidphenol:
chloroform(酚:
氯仿)
b.Vortexthoroughly.Centrifuge5 min,roomtemperatureattopspeedinamicrofuge(≥10,000 x g).Transferaqueousphase(toplayer)toanewtube.
c.Add75 μlchloroform(氯仿),vortexthoroughly,centrifuge5 mins.,roomtemperatureattopspeedinamicrofuge(≥10,000 x g).Transferaqueousphase(toplayer)toanewtube.
d.Add75 μlisopropanol(异丙醇),vortexthoroughly.Chillonicefor10 minutes.
e.Centrifugeatmaximumspeedfor20 minutes.Rinsepelletwith0.25 mlcold70%ethanol,centrifuge5 minutesatmaximumspeed,removeethanolcarefullyanddiscardit.Allowpellettoairdry.
f.Resuspendpelletin5.5 μlNuclease-freeWater.
g.PlacethemajorityofthesampleoniceandproceedtoTAPreaction,orstorethesampleat–20°C.
3.TobaccoAcidPyrophosphatase(TAP,烟草酸焦磷酸酶)treatment
a.AssemblethecomponentsinanRNase-freemicrocentrifugetube:
Amount(μl)Component
2.5μlCIP’dRNA(fromfstepabove)
0.5μl10×TAPbuffer
1μlTobaccoAcidPyrophosphatase
1μlNuclease-freeWater
b.Mixgently,spinbriefly.Incubateat37°Cforonehour.
c.Storereactionat–20°Corproceedtoligationstep.
4.[5' RACE]AdapterLigation
a.AssemblethecomponentsinanRNase-freemicrocentrifugetube:
Amount(μl)Component
1μlLigatedRNA
2μldNTPMix
1μlRandomDecamers
1μl10×RTBuffer
0.5μlRNaseInhibitor
0.5μlM-MLVReverseTranscriptase
To10μlNuclease-freeWater
b.Mixgently,spinbriefly.
c.Incubateat42°Cforonehour.
d.Storereactionat–20°CorproceedtothePCRstep.
5.NestedPCRfor5' RLM-RACE
The5' RACEOuterPrimerworkswellinPCRusinganannealingtemperaturefrom55to65°C.Therefore,anannealingtemperatureof60°Cisprobablyareasonablestartingpoint.Theoptimaltemperatureforyourprimerandtemplatecombinationmayhavetobedeterminedempirically.
[7]TOPOTACloningprotocol
1.ProducePCRproductsusingTappolymeraseandyourownprotocol.
2.AnalyzeeachPCRsamplebyagarosegelelectrophoresis.
3.Add:
FreshPCRproduct0.5to1μl
SaltSolution0.5μl
TOPOvector0.5μl
Wateraddtoatotalvolumeof3μl
4.Mixreactiongentlyandincubatefor5minutesatroomtemperature(22-23°C).(Afterthat,youmaystoretheTOPO®Cloningreactionat-20°Covernight).
5.Thaw(解冻)onice1vial(玻璃瓶)ofOneShot®cellsforeachtransformation.
6.Add3μloftheTOPO®CloningreactionintoavialofOneShot®E.colicellsandmixgently.Donotmixbypipettingupanddown.
7.Incubateonicefor5to30minutes.
8.Heat-shockthecellsfor30secondsat42°Cwithoutshaking.
9.Immediatelytransferthetubestoice.
10.Add250μlofroomtemperatureS.O.C.medium.
11.Capthetubesandshakeat37°Cfor1hour.
12.Sprea
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