Class Note for CHEM 490 at UMass(7)
Class Note for CHEM 490 at UMass(7)
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Date Created: 02/06/15
Recombinant DNA me39rhods Ch 5 Wed 21809 Biology does amazing chemis rry polymers of defined leng rh sequence unique s rruc rure reagen rs for experimen rs amp applica rions Making recombinan r DNA overview Res rric rion enzymes cu r DNA a r specific sequences Gel elec rrophoresis hybridiza rion Sou rhern blo r ring e rc DiA inser red in ro cloning vec ror Proper ries of cloning vec rors plasmids DiA is Transformed in ro hos r cells for amplifica rion Cell s machinery replica res DNA Se ec rionscreening for cells con raining rarge r DNA Libraries Genomic cDNA App ica rions Pro reome assign func rions ro genes Produce al rered plan rs or animals Produce biomolecules Pro rein expression amp si redirec red mu ragenesis Polymerase Chain Reac rion PCR Making recombinant DNA overview Restriction enzymes cut DNA at isolate genomic DNAand cleave C demmm h 31 ni2quot DNA Inserted Into clonIng vector restriztion enzyme whiff i Properties of cloning vectors plasmids gm 1 6 DNA is transformed into host cells liigls frs for amplification Llfml r W 50binam Cell s machinery replicates DNA ODNAm39ew39e Selectionscreening for cells 0 containing target DNA V I 4 a tell zrzmmz imp 39 I M VA ll 3 quotcsgl rproduceclones PAltvwquotl S 2006 Pearson Prenlke Hall Inc RWS39GJGltTCC 339 L I S L df bifk Pk i xbd PS i er squot 339 CCTAGG 539 BamHl T Restriction endonucleases i Restriction enzymes 539 GAATTC 339 7quot i I EcoRl S 339 CTTAAG 539 a T Wu 5 GGJ CC 339 H Ill 3 CCGG 539 ae T quotW r t i 5322 2 W T exonucleases l I r S M I 212212 3 1 Wm Figure 54 Wmm mm LN asg 1 I new H mm 13 b The endonuclease recognizes the Following DNA replication the Restriction G A A T T C Sequence Md CleaVes bOth G A A T T C site is hemimethylate strands of the foreign DNA to produce fragments with staggered ends 539vaNN 339 5 Nng39 339mNN 5 3 NNms39 Replication J Amethylase catalyzes ECORl blndS and Cleaves Methylation methylation of the second adenine residue in the recognition site 4 was Figure 1930 Principles of Biochemistry le 9 2005 Pearson Prentice Hall Inc EcoRI doesn t cleave methylated recognition sequence Figure 1931 Principles of Biochemistry Ale 9 2005 Pearson Prentice Hall Inc Major groove Major groove Minor groove Minor groove AdenineThymine Guanin ECylosine Major groove Major groove Minor groove Thymine Adenine Minor groove cytosine Guanine mu or graavz39 01M39 c Awwir c 9 09079 TWMA GAC pair AT um39r CC ymir TA pair L minor rm f r e 3 x r G C KEY A g G T 0 Hvoud mentor Himmi dmur c m G Iiydmgmldlom 0 nuU lyl grow T O O A l39gtO a maior groove minor groove GC pair G p C KEY AT pair A T O Hbond raptor A O Hbond donor C39C39 Wquot C G ydrogen atom TA pair T A Q met yl group 1 610quot GARLAND PUBLISHING INC A number oi the Tam39bv mm Gran Sma l T A C G G C C G G C C G C G G C C G G C A T G C GUN GAPLAND PUBLISHIVG INC A usva of me mm c mes Gum Gel dedm or s A39MM PW 2mg Q kmm Wid39NM mum ram Abel DNA fragment Transfer 7 Add DNA 39 7 31 probe 3 ofDNA Plabeled I d E by blotting 7 DNA probe Autoradiography revea E g r g 7 gt gt E 7 III E 7 Agnrose Nilwtellquse Autorudiogrum gel sheet sigmsa d WWWm siuhsdinun NewHimmmWW Southern DNA gel Northern RNA gel Western protein gel antibody Cleavage Re ognilion Cleavage site se uences site i G39AAIUAGETTC CATTAGCAG39CTGTAGC cquotquot quot 5 quot39 39 CTTAAGTCGAAG GTAMccTciGACATCG DNA T T EraRI Pvull remiction reminion endonuclease endonudease C quot 55 HWAGCTTCGCATTAGCAG cmmac cc TCGAAGCGTAATCGTC GACMCG Sticky ends w ends 139 Q MEM Mir 1m gl nh w 3 3m do Sa b Plagmid cloning vector cleaved with EcoRI and Pvull BamHl Sall Ampicillirk l Tetracycline resistance resistance ampR tetR pBR322 4361 bp drulm 1mm Y klo H P r replicabb aahv owl 39rA Vlw i39 11 64gt quot 5 at mix39lnkc 3 Origin of N r 49mm replication ori pvtuquot quothf e hb quotquot 39 K Swan s R pBR322 Q Q amp plasmids tetR D Q Q pBR322 is cleaved at the ampicillin l Pstl restriction re5stance element by Pstl endonuclease G a SQ 21 r i I Sall Q Q Foreign DNA MW 22312 1 h A S d Mustma Origin of replkation oril I Pvull Tetracycline 39 resistanu new Ampicillin resistante MP pBR322 436 lbp selection of transformed cells We All colonies A9quot rep ication I have plasmids containing on PWquot tetracycline IndIVIdual colonles are transferred to matching positions on additional NM S G plates One plate contains tetracycline colonies transferred we the other tetracycline and ampicillin for testin 9 8399 M l N 0 Colonies with wk e we 9 l s pQ recombinant o Sctcen check a cells kg i m v sh wl39 Sum plasmids 3990 Agar containing tetracycline control Agar containing ampicillin tetracycline Er no on Ho Cl HO H H 0 H o LGalacmsIdase 5r 0 OH on I gt N OH OH u H x433 1g URBAN 5bwmoLchloloShydroxylndole galactose 5mmm4cmumamunlyxpngalam Iannside Dxlda an Wkipedia U Bacterialpmmoterm Protein ExpreSSIon plasmid and operator 0 sequences Pol linker with unique Sites for several restriction endonucleases ie cloning sites Gene encoding repressor that binds O and regulates P Transcription termination sequence Ribosome binding site ori Selectable genetic marker eg antibiotic resistance mRNA a cDNA Mam a em 69 0e D L39lmr GENMK M VM MA mRNA 5 39 A A A A A A A A mRNA template is annealed to synthetic 39 oligonucleutide oligo dT primer 5 39 A A A A A A A A 3 39 T T T T T T T T Reverse transcriptase and dNTPs yield a complementary v DNA strand mRNADNA hybrid 5 39 A A A A A A A A 3 39 T T T T T T T T mRNA is degraded with alkali v 3 39 T T T T T T T T DNA polymerase I and dNTPs yield doublestranded DNA Duplex DNA 5 A A A A A A A A 3 39 T T T T T T T T Sample 1 Mix Sample 2 and cool Duplex of sample 1 Duplex of sample 2 Bacterial promoter site Euka ryuti DNA insert Expressinn liedar plasmid Transform E 0 4 Cnlony producing protein of interest In enul colonies on agar plule Transfer colonies to a replies plate Lyse bacteria to expose proteins Transfer proteins to nitrocellulase sheet Add radinlabeled antibody specific for protein of interest Darkspoton lm idem esthe bacterl coluny expressing the geneofinterest Auiorudiugrum Figure 53n Biathrml39stry 5ith Editian o zoow lureenonm colour WWQ DNA microarray DNA chip 18X180m CD w 392 39 39 helmmumsynmunx alllhegennslxumssldm he elis a IHM Stage gt132 x 02 Hiquot 4 oxwen mum to mm by mean quotan p Em r vmfsr using ucrescemlylaheled lumswmse dmxyribnnudemxd lriphosphams g n m um mm m micruway uuomsmm n DNA anneal m amplememary szquenles on Ihe nimnnay miunarray Removal o unnybrzdixed probe lt9 mm J reuvexenls a gene exuressed V 2 inlheullx De6 n gt r li nr obilized DNA 6200 genes of yeast
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