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        Yeast Gene knockouts using Oligo/PCR

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        Yeast Gene KO using Oligo/PCR

        Universal primers for gene knock-out using dominant drug markers: Kan, Clonat, and Hygromisin-B.

        Forward primer: 5’ TCAGGGGCATGATGTGACT 3’
        Reverse primer: 5’ AGCTCGTTTTCGACACTGGAT 3’

        Primers for hisG::URA3::hisG
        Forward primer 5" AGGGAACAAAAGCTGGGTAC 3"
        Reverse primer 5" CTATAGGGCGAATTGGAGCT 3"

        Plasmids for Selectable Markers
        Plasmid for amplifying Kan: pKan-GenMX4. seq (GCK), product size: ~1.4kb.
        Plasmid for amplifying Clonat: pAG25-ClonatMX4. seq (GCK), product size: ~1.2kb.
        Plasmid for amplifying HB: pAG32-hphMX4. seq (GCK), product size: ~1.5kb.
        Plasmid for amplifying hisG::URA3::hisG: pMPY-Zap seq (GCK) product size ~2.2kb

        PCR condition:
        1. 95°C for 5 minutes
        2. 95°C for 30 seconds
        3. 57°C for 1 minute
        4. 72°C for 3 minutes
        5. Repeat steps 2 to 4 for 30 cycles
        6. 72°C for 5 minutes
        7. 4°C Forever


        Primers for colony PCR to confirm gene knockout using Kan, Clonat, and HB.
        For Kan:
        Forward: KanMX4F1: 5’ ATTCTCACCGGATTCAGTCGT 3’
        Reserve: KanMXR1: 5’ AATCCGGTGAGAATGGCAAA 3’
        For Clonat:
        Reverse: NatMX4R1: 5’ ATTCGTCGTCCGATTCGT 3’
        For H-B:
        Forward: hphMX4F1: 5’ TACACAAATCGCCCGCAGAA 3’
        Reverse: hphMX4R1: 5’ TCGGTTTCAGGCAGGTCTT 3’


        You need to design a PCR primer for your gene of interest to make it work.

         

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