2 Hybrid System TRAFO Protocol_蚂蚁淘，【正品极速】生物医学科研用品轻松购|ebiomall -蚂蚁淘商城

商品列表技术文章相关问答

当前位置： > 首页 > 技术文章 >

2 Hybrid System TRAFO Protocol

来自 : 蚂蚁淘

Agatep,R.,R.D.Kirkpatrick,D.L.Parchaliuk,R.A.Woods,andR.D.Gietz(1998)TransformationofSaccharomycescerevisiaebythelithiumacetate/single-strandedcarrierDNA/polyethyleneglycol(LiAc/ss-DNA/PEG)protocol.TechnicalTipsOnline(http://tto.trends.com).

Forcompleteinstructionsonhowtodoatwohybridscreenseethefollowingreferences.

1.Gietz,R.D.,B.Triggs-Raine,A.Robbins,K.C.Graham,andR.A.Woods(1997)Identificationofproteinsthatinteractwithaproteinofinterest:Applicationsoftheyeasttwo-hybridsystem.MolCellBiochem172:67-79.

2.Parchaliuk,D.L.,R.D.Kirkpatrick,R.Agatep,S.L.SimonandR.D.Gietz(1999)Yeasttwo-hybridsystemscreening.TechnicalTipsOnline(http://tto.trends.com)(accepted).notonlineyet

SEETRAFOREFs

ThisTRAFOProtocolisfor2Hybridsystemscreens(Wehavedoneover30andstillcounting)

HighEfficiencyTransformationofayeaststrainrequiringmaintenanceofaplasmid.

Thetwo-hybridsystemandothersimilargeneticscreensinyeastinvolvetheuseoftwodifferentplasmidsinasingleyeastcell.OneplasmidoftencontainsaclonedgeneorDNAsequencewhiletheotherplasmidcontainsalibraryofgenomicorCDNA.Whilebothplasmidscanbeco-transformedintoasingleyeastcell,itisoftenmoreefficienttotransformthelibrarypoolintoastrainalreadycontainingthefirstplasmid(R.D.Gietz,unpublisheddata).

Toprepareaplasmid-carryingstrainforanadditionaltransformation,theinitialgrowthphaseshouldbeusingSComissionmediauntilthecelltiterreaches1-2x10⁷cells/ml.Thiswillmaintainthefirstplasmidduringthisgrowthphase.ThecellscanthenbesubculturedinYPADmediumfortwodoublingswithoutsignificantplasmidloss,iftheplasmidhasnoeffectonyeastgrowth.Toproducehighlycompetentyeastcellscontainingaselectableplasmid,followtheprotocolbelow.TodeterminetheScaleupneededforthedesirednumberoftransformants,werecommendtestingone"slibraryDNAwiththeprotocolbelowtodeterminethenumberofTransformantsthatcanbeexpected.Thenselecttheconditionsthatgivea"good"Transformationyieldandthenscaleupto30X,60Xoreven120Xobtainthedesirednumberoftransformants.(seeTable1&Discussion).

AllsolutionsusedinthisprotocolaredescribedintheTRAFOSolutionsPage

InoculatetheyeaststraincontainingthefirstplasmidintotheappropriatevolumeoftheappropriateSC-omissionmediuminaflaskandincubateat30^oCovernight.
Determinethecelltiterandcalculatethevolumeofcellsthatyields2.5x10⁸cellsforeach50mlsofYPADcultureneeded
Pourthisculturevolumeintoanappropriatesterilecentrifugetubeandpelletthecellsat3000xgfor5min.ResUSPendthecellpelletintheappropriatevolumeofpre-warmed(30^oC)YPADandtransfertoanothersterilecultureflask.
Incubateat30^oCwhileshakingat200rpmfor3to4hrsuntilthecelltiterreaches2x10⁷cells/ml.
Harvestthecellsbycentrifugationat3000xgfor5min.
Washthecellpelletviaresuspensionwith1/2volumeofsddwaterandcollectionbycentrifugationasabove.
Resuspendthepelletintheappropriatevolumeof100mMsterileLiAcandtransfertoanappropriatecentrifugetube.Incubatefor15minat30^oC.Pelletthecellsagainbycentrifugationandremovethesupernatant.
1. Add,intheorderfromtoptobottom,thecomponentsofthetransformationmixlistedinthetablebelowtoaseperatetubeandmixthoroughlybyvortexing.Addthetransformationmixtothecellpelletandvortexvigorouslytoresuspendthecellpellet.AlternativelyyoumayalsomixallcomponentsbuttheplasmidDNAtogether.AddtheentirevolumeofthetransformationmixminustheplasmidDNAtothecellpelletandthenaddtheplasmidDNAandmix.ThiswillkeepyoufromloosinganyplasmidDNAwhentransferingtheviscousliquidofthetransformationmixontopofthecells.
  Pleasenote:Thevaluesforeachscaleupshouldbemultipliedfromthesinglereactionvolumes.Previouslythe60XscaleupvaluesfortheLiAc,SS-DNA,andPlasmidDNAwereNOTcorrect!(Theywere90Xscale,sorry)ThenumbersshownhereareNOWcorrect!Thankstothepersonthatcaughtmyerrorandsorrytoallofyoubattlingtogetgood2HSscreensdone.Inaddition,Pleasenotethatwearenowadding2Xtheamountofcarrierthaninpreviousversionsofthisthispage.TIP:
  - Vigourouslyvortexthecellpelletuntilitistotallyresuspended,whichshouldtakeabout1min.Ifyouhaveproblemsgettingthepelletresusupendedletitsitfor5minandthenvortex!
  - Incubatethetransformationmixat30^oCfor30min.
  - Heatshockat42^oCfortimeindicatedbytablebelowwithmixingbyinversionfor15secafterevery5min.
    TIP:
  - Collectthecellsbycentrifugationasabove.GentlyresuspendthecellpelletinanappropriatevolumeofsddwaterandplateontoSCommissionmedium.Forour30Xand60X2hybridscreensweplateonto100largeplates.(Yes!thatiscorrect!Awholecaseoflargeplates)Thisgivesbettertransformationandlibrarycoverage!Forthe10Xscreensweusedless.
    TIP:
  - Incubatetheplatesfor3-5daysat30^oCoruntilcoloniesappear.ForsometwohybridScreenwewaitaslongas14days!
    Discussion
    ItisimportanttooptimizetheamountoflibraryplasmidDNAforeachstandardtransformation.AsshowninTable1below,suchatestisdonebytransformingincreasingamountsoflibraryplasmidDNAinastandardtransformationreaction.Fromthisexperiment,onecanseethatitismoreproductivetodo10standardtransformationreactionswith1µgorplasmidDNA(orscaleupthetransformationreaction)thantodoonestandardtransformationusing10µgofplasmid.ThiswillnotonlyensureefficientuselibraryDNAbutwillalsoreducesthenumberofyeastcoloniescontainingmorethanonelibraryplasmid,whichcanmakesubsequentanalysisdifficult.
    Co-transformationoflibraryplasmidDNAwithaGAL4_BDplasmidisnotasefficientastransformationofthelibraryDNAintoayeaststrainalreadycontainingtheGAL4_BDplasmid.Therefore,itisrecommendedthattheGAL4_BDplasmidbetransformedfirstintotheyeaststrainusingtheQuick&Easyprotocolandfollowedbytransformationofthelibrarywithprotocolabove;wehavescreenedasmanyas5.2x10⁷transformantsfromasinglescaleduptransformationreactioninatwo-hybridscreen(R.D.Gietz,unpublisheddata).IncasesweretheGAL4_BDfusionplasmidaffectsthegrowthoftheyeaststrain,itmaybeadvisabletoco-transformtheGAL4_BDfusionplasmidandthelibraryplasmidDNA.Inthesecases,carefulattentiontothelevelsofeachplasmidinthetransformationreactionisnecessaryfortheproductionofthehighestefficiency.

免责声明本文仅代表作者个人观点，与本网无关。其创作性以及文中陈述文字和内容未经本站证实，对本文以及其中全部或者部分内容、文字的真实性、完整性、及时性本站不做任何保证或承诺，请读者仅作参考，并请自行核实相关内容。

发布于： 2018-12-26 阅读（170）

下一篇 : 如何与「心机」boy 和睦相处？实验室离心机安全必备知识

▍ 相关分类

基因编辑

CRISPR基因敲除转染试剂 CRISPR 细胞株 RNA干扰

克隆和表达

克隆试剂盒克隆基因基因突变双杂交系统转录表达

分子诊断

定量PCR 数字PCR系统恒温扩增

二代测序

簇生成和测序试剂靶向测序试剂盒 DNA文库制备试剂盒测序酶 RNA文库制备试剂盒核酸常温运输

核酸酶类

DNA聚合酶 RNA聚合酶 DNA修饰酶 RNA修饰酶连接酶限制性内切酶反转录酶其他

核酸提取纯化

DNA提取纯化试剂 RNA提取纯化试剂

其他分子产品

载体文库 Marker 标准品寡聚核酸外泌体研究引物与探针酶抑制剂其他合成

▍ 相关文章

more+

扫描电镜生物标本制备实验实验方法想要做小鼠的活体成像,应该怎样做最好？动植物与海洋生物丁香... 怎样去除14丁二胺双盐酸的盐酸盐有机学术科研... tulip biolabs上海代理品牌：tulip biolabs上海代理欧、美盖 ... 八臂迷宫实验方法请教小鼠结肠切片的问题猪低分子肝素(LMWH)elisa酶联免疫试剂盒报价_猪低分子肝素,LMWH... 日立推出全新荧光分光光度计F4700 完善荧光产品线分析测试... 细胞生物学实验思考题一种新型电导率可控的导电织物芳香化酶抑制剂相关骨关节痛治疗研究进展 RACE(rapidamplification_of_cDNA_ends)

▍ 相关问答

more+

TRAFOSCALE	10X	30X	60X
CultureSize	25mls	50mls	100mls

TRAFOSCALE	10X	30X	60X
YPADcultureSize	50mls	150mls	300mls
#ofCellsneeded	2.5x10⁸	7.5x10⁸	1.5x10⁹

TRAFOSCALE	10X	30X	60X
YPADcultureSize	50mls	150mls	300mls

TRAFOSCALE	10X	30X	60X
100mMLiAc	3mls	3mls	6mls

TRAFOSCALE	10X	30X	60X
50%PEG	2.4ml	7.20ml	14.40ml
1.0MLiAc	360µl	1.08ml	2.16ml
SS-DNA(2mg/ml)	500µl	1.50ml	3.00ml
LibraryplasmidDNA	Aµl	Bµl	Cµl
sddWater	340-Aµl	1.02-Bml	2.04-Cml

TRAFOSCALE	10X	30X	60X
HeatshockTime	30min	40min	45-60min

TRAFOSCALE	10X	30X	60X
ResuspensionVolume	10mls	40mls	40mls