BCH 5413 Module 2 Lecture 5: Cloning I Questions And Answers

BCH 5413 Module 2 Lecture 5: Cloning I
Questions And Answers
a.tAdditionaltfeaturestneededtintatyeasttshuttletvectortandtwhy?t-tCORRECTttANSWER-
Atyeasttshuttletvectortrequirestatyeasttorigintoftreplicationt(e.g.,tARS)tfortplasmidtreplicationt
andtatselectabletmarkert(e.g.,tURA3tortLEU2)tforttransformationtscreening.tAtcentromeretse
quencet(CEN)tmaytbetincludedtfortstabletplasmidtmaintenance

a.tBlunttendstgivestbettertligationtthantstickytendst-tCORRECTttANSWER-
FALSE.tStickytendstligatetmoretefficientlytthantblunttendstduettotcomplementarytoverhangst
thattaidtstability

a.tPolylinkert(MCS):t-tCORRECTttANSWER-
ProvidestmultipletrestrictiontsitestfortflexibletDNAtinsertion.

a.tTwotwaystcoloniestcantappeartontat"Notligaset/tNotinsert"tcontroltplate:t-
tCORRECTttANSWER--

tThetplasmidtwastnottcompletelytdigestedtandtremainedtcircular,tallowingttransformation
t

•tThetplasmidtself-ligatedtwithouttantinsert.

b.tAdditionaltfeaturestneededtintatmammaliantshuttletvectortandtwhy?t-
tCORRECTttANSWER-

Atmammaliantshuttletvectortneedstatstrongtpromotert(e.g.,tCMV)tfortgenetexpression,tatpoly
adenylationtsignaltfortmRNAtprocessing,tandtatselectabletmarkert(e.g.,tNeoR)tfortstabletcellt
selection.

b.tAntibiotictresistancetgene:t-tCORRECTttANSWER-
Allowstselectiontofttransformedtbacteriatbytenablingtgrowthtintantibiotictmedia.

b.tIfttheretaretmoretcoloniestontthetNotligaset/tNotinserttcontroltthantontthetexperimentaltplate
,tistthistgoodtortbad?tWhy?t-tCORRECTttANSWER-
Thistsuggeststincompletetdigestiontoftthetplasmid,tmeaningtatlargetnumbertoftundigestedtort
self-
ligatedtplasmidstarettransforming,tmakingtittharderttotobtaintthetdesiredtrecombinanttplasmi
d.