Labprotocols分子生物学实验方法技术汇总.docx

Labprotocols分子生物学实验方法技术汇总.docx

《Labprotocols分子生物学实验方法技术汇总.docx》由会员分享，可在线阅读，更多相关《Labprotocols分子生物学实验方法技术汇总.docx（88页珍藏版）》请在冰点文库上搜索。

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