Universalprimersforgeneknock-outusingdominantdrugMarkers:Kan,Clonat,andHygromisin-B.Forwardprimer:5’TCAGGGGCATGATGTGACT3’Reverseprimer:5’AGCTCGTTTTCGACACTGGAT3’PlasmidsforSelectableMarkersPlasmidforamplifyingKan:pKan-GenMX4.seq(GCK),productsize:~1.4kb.PlasmidforamplifyingClonat:pAG25-ClonatMX4.seq(GCK),productsize:~1.2kb.PlasmidforamplifyingHB:pAG32-hphMX4.seq(GCK),productsize:~1.5kb.PCRcondition:1.95°Cfor5minutes2.95°Cfor30seconds3.57°Cfor1minute4.72°Cfor3minutes5.Repeatsteps2to4for30cycles6.72°Cfor5minutes7.4°CForeverPrimersforcolonyPCRtoconfirmgeneknockoutusingKan,Clonat,andHB.ForKan:Forward:KanMX4F1:5’ATTCTCACCGGATTCAGTCGT3’Reserve:KanMXR1:5’AATCCGGTGAGAATGGCAAA3’ForClonat:Reverse:NatMX4R1:5’ATTCGTCGTCCGATTCGT3’ForH-B:Forward:hphMX4F1:5’TACACAAATCGCCCGCAGAA3’Reverse:hphMX4R1:5’TCGGTTTCAGGCAGGTCTT3’YouneedtodesignaPCRprimerforyourgeneofinteresttomakeitwork.
