分子生物学实验技术总结.ppt

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1、Techniques SummaryLi Xiangyun2011-3-28Select appropriate vector Get the target DNA sequencePCR primer designObtain cell or tissuePCR amplification Restriction,ligation and transformationScreen positive cloneSequencingVector construction procedureSelect appropriate vector Human cytomegalovirus(CMV)im

2、mediate-early promoter/enhancer:Permits efficient,high-level expression of your recombinant protein T7 promoter/priming site:Allows for in vitro transcription in the sense orientation and sequencing through the insert Multiple cloning site in forward or reverse orientation:Allows insertion of your g

3、ene and facilitates cloning Neomycin resistance gene:Selection of stable transfectants in mammalian cells pUC origin:High-copy number replication and growth in E.coli Ampicillin resistance gene(-lactamase):Selection of vector in E.coliGet the target DNA sequence http:/en.wikipedia.org/wiki/Main_Page

4、 http:/ atggcggtccgagcttcgttcgagaacaactgtgagatcggctgctttgccaagctcaccaacacctactgtctggtagcgatcggaggctcagagaacttctacagtgtgttcgagggcgagctctccgataccatccccgtggtgcacgcgtctatcgccggctgccgcatcatcgggcgcatgtgtgtggggaacaggcacggtctcctggtacccaacaataccaccgaccaggagctgcaacacattcgcaacagcctcccagacacagtgcagattaggcgggtgg

5、aggagcggctctcagccttgggcaatgtcaccacctgcaatgactacgtggccttggtccacccagacttggacagggagacagaagaaattctggcagatgtgctcaaggtggaagtcttcagacagacagtggccgaccaggtgctagtaggaagctactgtgtcttcagcaatcagggagggctggtgcatcccaagacttcaattgaagaccaggatgagctgtcctctcttcttcaagtcccccttgtggcggggactgtgaaccgaggcagtgaggtgattgctgctgggatgg

6、tggtgaatgactggtgtgccttctgtggcctggacacaaccagcacagagctgtcagtggtggagagtgtcttcaagctgaatgaagcccagcctagcaccattgccaccagcatgcgggattccctcattgacagcctcacctgagtcaccttccaagttgttccatgggctcctggctctggactgtggccaaccttctccacattccgcccaatctgtaccggatgctggcagggaggtggcagagagctcactgggactgaggggctgggcacccaacccttttccacctgtgctt

7、atcgcctggatctatcattactgcaaaaacctgctctgttgtgctggctggcaggccctgtggctgctggctgagggttctgctgtcctgtgccaccccattaaagtgcagttccctccggaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa MAVRASFENNCEIGCFAKLTNTYCLVAIGGSENFYSVFEGELSDTIPVVHASIAGCRIIGRMCVGNRHGLLVPNNTTDQELQHIRNSLPDTVQIRRVEERLSALGNVTTCNDYVALVHPDLDRETEEILADVLKVEVFRQTVADQVLV

8、GSYCVFSNQGGLVHPKTSIEDQDELSSLLQVPLVAGTVNRGSEVIAAGMVVNDWCAFCGLDTTSTELSVVESVFKLNEAQPSTIATSMRDSLIDSLT PCR primer designAAGCTTATGGCGGTCCGAGCTTCGTTC CCCTGTGGCTGCTGGCTAG GGGACACCGACGACCGATCAGATCTHindXbau The target gene doesnt contain corresponding restriction sites.u Tm：(G+C)4+(A+T)2=6070u Correct reading

9、 frameu Based protectionPCR amplificationTaq MasterMix,2Forward primer(10M)Reverse primer(10M)TemplateRNase-Free waterTotal25l2l2l1gUp to 50l50lFinal conc.10.4M0.4MInitial denaturationDenaturationAnnealingExtentionFinal extention949455-6572722min30s30s30-90s5-10min25-35cyclesPCR reaction systemsPCR

10、cyclingTaq DNA Polymerase:Misincorporation identificationTaKaRa Ex Taq:35 Exonuclease target genes amplificationTaKaRa Hot Start Taq:affibody decrease non-specific amplificationRestriction,ligation and transformation1g DNA:1020U enzymeStar activity:enzyme volume/total volume 1/10Buffer 10TangoTMHind

11、XbaddH2OTotal5l4l2lUp to 50l50l37,2hAATGCCATTCATTACGGTAAG GAACTGCAGTCTTGACGTCAATGCCATTCAGAACTGCAGTTACGGTAAGTCTTGACGTCT4 DNA LigasePCR productT vectorVector 5lTarget gene 5lSolution 10lVector 1lTarget gene 1lddH2O 3LSolution 5lVector:Target gene=1:210Calcium chloride(CaCl2)transformation co-immunopre

12、cipitation Cell-Free Expression Systems The most frequently used cell-free translation systems consist of extracts from rabbit reticulocytes,wheat germ and Escherichia coli.All are prepared as crude extracts containing all the macromolecular components(70S or 80S ribosomes,tRNAs,aminoacyl-tRNA synth

13、etases,initiation,elongation and termination factors,etc.)required for translation of exogenous RNA.To ensure efficient translation,each extract must be supplemented with amino acids,energy sources(ATP,GTP),energy regenerating systems(creatine phosphate and creatine phosphokinase for eukaryotic syst

14、ems,and phosphoenol pyruvate and pyruvate kinase for the E.coli lysate),and other co-factors(Mg2+,K+,etc.).There are two approaches to in vitro protein synthesis based on the starting genetic material:RNA or DNA.Standard translation systems,such as reticulocyte lysates and wheat germ extracts,use RN

15、A as a template;whereas coupled and linked systems start with DNA templates,which are transcribed into RNA then translated.Rabbit Reticulocyte LysateIn standard translation reactions,purified RNA is used as a template for translation.“Linked”and“coupled”systems,on the other hand,use DNA as a templat

16、e.RNA is transcribed from the DNA and subsequently translated without any purification.Such systems typically combine a prokaryotic phage RNA polymerase and promoter(T7,T3,or SP6)with eukaryotic or prokaryotic extracts to synthesize proteins from exogenous DNA templates.DNA templates for transcripti

17、on:translation reactions may be cloned into plasmid vectors or generated by PCR.Linked Transcription:Translation two-step reaction,based on transcription with a bacteriophage polymerase followed by translation in the rabbit reticulocyte lysate or wheat germ lysate Coupled Transcription:Translation o

18、ne-step reaction,during transcription,the 5 end of the RNA becomes available for ribosomal binding and undergoes translation while its 3 end is still being transcribed.TNT Rabbit Reticulocyte LysateAmino Acids Minus MethionineAmino Acids Minus LeucineTNT Reaction BufferTNT T7 PolymeraseRNasin Ribonuclease InhibitorTarget DNAddH2OTotal13l0.25l0.25l1l0.5l1l1gUp to 25l25l30,90min2l for WBIn vitro Sumoylation-K-x-D/E maturationE1 enzymeSUMO-2Reaction bufferUbcH9 enzymeTarget proteinddH2OControl group:NO ATPExperimental group:Mg-ATP solutiontotal2l2l2l2l2lUp to 18l2l2l20l