5 - Molecular Cloning - The professor\'s name was Jeff Laney. This is for exam one. PDF

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The professor's name was Jeff Laney. This is for exam one....

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Sunday ,May2,y

MCB304 Molecular Cloning

-Rest r i ct i onenzymes •Recogni z e48bas epai rl ongpal i ndr omi cs equences •Cancausest i ckyends( endsofeachDNAs t r andst i ckout )orbl untends( enzyme cut sst r ai ghtt hr oughbot hst r ands)

•Di gest :cutapar tasampl eofDNAusi ngr est r i ct i onenzymes •Rest r i ct i onf r agment :pi eceofDNAt hatr esul t sf r om di gest i ngal ar gers ampl e •Compl et edi gest :di gestwher eever ypossi bl ecut t i ngsi t ei sc l eav ed( cont r asti sa par t i aldi gest )

•Rest r i ct i onmap:mapofar egi onofDNAwi t hr est r i ct i onsi t esandt hedi st ancesbet weent hem ( i nkb)mar k ed)

-Runni nggel s •Si z esoff r agment scanbedet er mi nedusi nggel el ect r ophor esi s -Makeagelwi t hwel l st ocont ai nDNA -Pl acegeli nbuffer edsol ut i onandat t achacur r ent •Negat i v eel ect r odei sat t achedt ot heendwi t ht hewel l sbecauseDNAhasa negat i v echar ge

-DNAi sl oadedi nt hewel l sandwi l lmi gr at et owar dst hepos i t i v eel ect r ode -Geli sst ai nedwi t het hi di um br omi deandphot ogr aphedunderUVl i ght -Si z ei sdet er mi nedbyc ompar i sont ot hemi gr at i onofaknownmar k er •Smal l erDNAf r agment sar el esshi nder edbyt hegelandwi l l mi gr at ef ur t her -Mappi ng 1

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•Definedl i nearDNAsgi veanumberoff r agment sequalt ot henumberofcut spl us one

•Ci r cul arDNAsgi v eanumberoff r agment sequalt ot henumberofcut s •Fr odefinedDNAs ,t hesum ofal l f r agmentl engt hsi neachdi gestwi l lbet hes ame •I nadoubl edi gest ,af r agmentt hati st hesamesi z easonei nasi ngl edi gestmi ght bet hesamef r agmentoranewf r agment

•Eachf r agmenti nadoubl edi gesthadt ocomef r om af r agmenti neachoft hesi ngl e di gest st hatwasatl eastasl ar geast hedoubl edi gestf r agment

•Onaver age,r est r i ct i onenzymescutev er y4^ nbasepai r s( n=l engt hofr ec ogni z ed sequence)

-Canr educ et hec ompl exi t ybyPCRormol ec ul arcl oni ng -Mol ecul arc l oni ng:amol ecul ar ,genomewi desear chandr ecov er ymi ssi onf orapar t i cul ars equence

•Fi ndDNAt hatcont ai nsf r agment sofi nt er est -I ns er tt hef r agment si nt ov ect or s( gener at er ecombi nantDNAmol ecul es) -Tr ans f erv ect or i nser thybr i dmol ec ul esi nt ocel l ssot hecel l sampl i f yt hesemol ecul es( r epl i cat er ecombi nantDNAmol ec ul es)

•Cel ldi vi si onr esul t si nbi l l i onsofi dent i calcopi es( cl ones)oft hei nser t edf r agment •Af t erdi gest i onwi t hr est r i ct i onenzymes,DNAf r agment scanbeputbackt oget her andr eseal edwi t hDNAl i gas e

•St eps -I sol at eami xt ur eofDNAf r agment sf r om anor gani sm’ sgenome -I ns er tpi ecesofDNAi nt ocl oni ngvect or st ocr eat eal i br ar y( col l ec t i onofc l oned DNAf r agment sr epr esent i nganor gani sm’ sgenome)

•Genomi cl i br ar y :col l ect i onofcl onedDNAf r agment sr epr esent i ngt heent i r e genomeofanor gani sm 2

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•cDNAl i br ar y :col l ect i onofcl onedDNAf r agment sr epr esent i ngonl yi nf or mat i on f oundi nmRNAt r anscr i pt sofanor gani sm ( DNAcopi esofmRNA)andcan car r yi nf or mat i onaboutt het ypesofmRNAf oundi ndi ffer entcel l soft hebody

-Scr eenl i br ar yf orgenes/ mar k er sofi nt er est •Hybr i di zat i on -I dent i f yDNAf orpar t i cul argenesandi sol at ei tf r om ac ompl expi ct ur e -CanuseDNAswehavet opr obef ormor eDNAst hatar et hesameorsi mi l ar •Pr obecanbeal r eadycl onedDNAf r om or gani sm,homol ogousgenef r om anot heror gani sm,orDNAchemi cal l ys y nt hesi z edf r om aknownsequence

-I nsi t u •Fi ndsspec i fics equencesi nmor ecompl exmi xt ur es •Bl ot t i ng -Sout her n( DNA)i nv ent edbyEdwi nSout her n •St eps -Denat ur eDNAandt r ansf ert ofil t erpaper -Putfil t eri nbagwi t hl abel edDNApr obes -Pr obeshybr i di z et ocompl ement ar ysequences •Det er mi neshowmanydosesofagenear epr esent •Looksf orc hangesi ngenest r uct ur e( i nv er si on,del et i on,t r ansl ocat i on,i nser t i on)

•Scr eensf orsi l entgenet i cvar i ant s( r est r i ct i onsi t es) -Nor t her n( RNA) •Det er mi nesi fRNAi sexpr essedi napar t i cul art i ssueandt hel evel ofexpr essi on

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Sunday ,May2,y

•Looksf oral t er nat i v esi z ef or ms •Looksf orc hangesi nexpr essi onl ev elwi t hc hangesi not hergenes -West er n( pr ot ei ns) •Usesant i bodi est odet ectpr ot ei nofi nt er est

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