Day2

1.Determinethetiteroftheyeastculturebypipetting10mlofcellsinto1.0mlofwaterinaspectrophotometercuvetteandmeasuringtheODat600nm.FormanyyeaststrainsasUSPensioncontaining1x10⁶cells/mlwillgiveanOD600of0.1.Alternatively,titerthecultureusingahemocytometer.seenote:

---

Note:

i)DiluteovernightYPADorSCcultures10^-1ormoreinwater.

ii)Carefullyplace10µlofthecellsuspensionbetweenthecoverslipandthebaseofhaemocytometer.Letthecellssettleontothehaemocytometergridforafewminutes.Thegridareaistypically1squaremillimeter,dividedinto25equal-sizedsquares,andthevolumemeasuredis10^-4ml.

ii)Countthenumberofcellsin5diagonalsquares

iv)Calculatethecelltiterasfollows:cellscountedx5xdilutionfactorx1/volumemeasuredbythe25squaresofthehaemocytometer.239cellsx5x10(dilutionfactor)x1/10^-4ml=1.2x10⁸cells/ml.

v)Saccharomycescerevisiaedividesbybuddingfromamothercell.Countbuddedcellsasasinglecells.Countcellswithequalbudsizesastwocellswhenthereisevidenceofadditionalbudsformingoneithercell.Somestrainsformclumpsofcellswhichreduceplatingefficiency.Asingleclumpofcellswillonlygiverisetoonecolonyonaplate,whichmaycomplicatefurtheranalysis.

---

2.Transfer50mlofthepre-warmed2xYPADtothepre-warmedcultureflaskandadd2.5x10⁸cellstogive5x10⁶cells/ml.

3.Incubatetheflaskonarotaryorreciprocatingshakerat30°Cand200rpm.

Note:

i)Itisimportanttoallowthecellstocompleteatleasttwodivisions.

ii)Thiswilltake3to5hours.

iii)Thisculturewillgivesufficientcellsfor10transformations.iv)Transformationefficiency(transformants/µgplasmid/10⁸cells)remainsconstantfor3to4celldivisions.

4.Whenthecelltiterisatleast2x10⁷cells/ml,whichshouldtakeabout4hours,harvestthecellsbycentrifugationat3000gfor5min,washthecellsin25mlofsterilewaterandresuspendin1mlofsterilewater.

5.Boila1.0mlsampleofcarrierDNAfor5minandchillinanice/waterbathwhileharvestingthecells.

****ItisnotnecessaryordesirabletoboilthecarrierDNAeverytime.Keepasmallaliquotinyourownfreezerboxandboilafter3-4freeze-thaws.Butkeeponicewhenout.****

6.Transferthecellsuspensiontoa1.5mlmicrocentrifugetube,centrifugefor30secanddiscardthesupernatant.

7.Addwatertoafinalvolumeof1.0mlandvortexmixvigorouslytoresuspendthecells.

Note:Ifthecelltiterofthecultureisgreaterthan2x10⁷cells/mlthevolumethenincreasethevolumetomaintainthetiterofthissuspensionat2x10⁹cells/ml.Ifthetiterofthecultureislessthan2x10⁷cells/mlthendecreasevolume.

8.Pipette100µlsamples(ca.10⁸cells)into1.5mlmicrofugetubes,oneforeachtransformation,centrifugeattopspeedfor30secandremovethesupernatant.

9.MakeupsufficientTransformationMixfortheplannednumberoftransformationsplusoneextra.KeeptheTransformationMixinice/water.

10.Add360µlofTransformationMixtoeachtransformationtubeandresuspendthecellsbyvortexmixingvigorously.

11.Incubatethetubesina42°Cwaterbathfor40min.

Note:Theoptimumtimecanvaryfordifferentyeaststrains.Pleasetestthisifyouneedhighefficiencyfromyourtransformations.

12.Microcentrifugeattopspeedfor30secandremovetheTransformationMixwithamicropipettor.

13.Pipette1.0mlofsterilewaterintoeachtube;stirthepelletbywithamicropipettetipandvortex.

Note:WeliketobeagentleaspossIBLeatthisstepifhighefficiencyisimportant.Excessivewashingwashesawaytransformants!!!!

14.PlateappropriatedilutionsofthecellsuspensionontoSCselectionmedium.Fortransformationwithanintegratingplasmid(YIp),linearconstructoroligonucleotide,plate200µlontoeachof5plates;foraYEp,YRporYCplibraryplasmiddilute10µlofthesuspensioninto1.0µlofwaterandplate10and100µlsamplesontotwoplateseach.The10µlsamplesshouldbepipetteddirectlyinto100µlpuddlesofsterilewaterontheSCselectionmedium.

Note:WhenspreADIngyeastinoculumontotheplategentlydistributethefluidcompletelywithasterileglassrodwithaminimumofstrokes.Allowthefluidtobetakenupbytheplatepriortoincubation.

15.Incubatetheplatesat30°Cfor3to4daysandcountthenumberoftransformants.Thetransformationefficiency(transformants/1µgplasmid/10⁸cells)canbedeterminedbycalculatingthenumberoftransformantsin1.0mlofresuspendedcellsper1.0microgramplasmidper10⁸cells.Forexample,ifthetransformationof1.0x10⁸cellswith100nanogramplasmidresultedin500coloniesonaplateofSCdropoutmediumspreadwith1µlofsuspension(usuallydispensedintoa100µlpuddleofsterilewaterontheplate).TransformationEfficiency=500x1000(platingfactor)x10(plasmidfactor)x1(cells/transformationx10⁸).TransformationEfficiency=5x10⁶transformants/1.0µgplasmid/10⁸cells.Transformationefficiencydeclinesasplasmidconcentrationisincreased(Gietzetal.1995)buttheactualyieldoftransformantspertransformationincreases.Forexample,100nanogramofplasmidinatransformationmightgiveaTransformationEfficiencyof5x10⁶andayieldof5x10⁵transformantswhereaswith1µgofplasmidtheTransformationEfficiencymightbe2x10⁶andtheyield2x10⁶pertransformation.Inordertoobtain5x10⁶transformantsitissimplertosetuptwoorthreetransformationswith1µgofplasmidDNA,orasingle3foldscaleduptransformation,thantocarryout10reactionswith100ngofplasmidineach.

Note:Twoplasmids,suchasanexpressionplasmidandalibraryplasmidpool,canbeco-transformedintoasinglecellbyincludingbothplasmidsinthesametransformationreaction.Theefficiencyisreduced,however,becauseonlyabout30-40%ofalltransformedcellstakeupmorethanoneplasmidmolecule.Analternativeapproach,whichhasahigherefficiencyistotransformintheexpressionplasmidfirst,usingthisortheQuickandEasyprotocol,andthenusetheprotocolfoundonthe2HybridSystemTRAFOPagetotransforminthelibraryplasmidpool.

================  蚂蚁淘在线  ================

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

版权声明：未经蚂蚁淘在线授权不得转载、摘编或利用其他方式使用上述作品。已经经本网授权使用作品的，应该授权范围内使用，并注明“来源：蚂蚁淘在线”。违反上述声明者，本网将追究其相关法律责任。

相关阅读

兔血管紧张素Ⅱ(ANGⅡ)elisa检测...

血小板表面及血浆内活化标记蛋白GMP14...

MICROFIL®血管造影剂品牌:Flo...

细说数码变焦—不很懂谈摄影基础知识 数...

关于我们 ICA 集团

Nature:新型抗生素或可解决细菌耐药...

蛋白上样缓冲液详解

Október 23a eml...

...shooting' mi...

质粒载体介绍(质粒基本特性和种类及标记基...

荧光定量pcr一定要做标准曲线吗

首页学而思官方网站

• 

  资质认证

  获得国家资质，权威认证！

• 

  全国联保

  全国联保，官方无忧售后

• 

  正规发票

  正规发票，放心购买

• 

  签订合同

  签订合同，保障您的权益

购物指南

购物流程

注册登录

账户管理

订单查看

常见问题

FAQS

蚁豆兑换

物流说明

评论规则

发票说明

价格说明

支付说明

服务协议

版权声明

诚信保障

免责申明

销售协议

隐私条款

法律申明

售后服务

退换货政策

工作时间

退款说明

售后支持

售后申请表模板列表

关于我们

公司简介

联系我们

资质证书

人才招聘

商务合作

万众创业

网址导航

蚂蚁淘商城

科研狗

苏州蚂蚁淘

生物信息网

现货促销


微信公众号

Calbiotech产品 | 转染试剂24765 | One Lambda价格 | Polysciences 商品规格 | biocorepl华东代理 | GeneTex超强WB发光底物 | SBA代理 | 蚂蚁淘 | 普通化学试剂

copyright©2005-2021 蚂蚁淘在线版权所有　苏州蚂蚁淘生物科技有限公司 www.assay-online.com ICP号：苏ICP备17049038号-16

/**/

NumberofTransformations
Reagents	1	5(6X)	10(11X)
PEG350050%w/v	240µl	1440µl	2640µl
LiAc1.0M	36µl	216µl	396µl
BoiledSS-carrierDNA	50µl	300µl	550µl
PlasmidDNAplusWater	34µl	204µl	374µl
Total	360µl	2160µl	3960µl