gene deletion & gene fusion protocol for yeast

Peccoud Lab Protocols: using PCR to delete a yeast gene or fuse a tag to a gene

Introduction

Yeast gene deletions or gene fusions (e.g. ORF with epitope tags or FP, promoter replacement, MS2 mRNA tagging) can be generated simply by transformation into yeast of a PCR amplicon containing the desired selectable marker or tag flanked by 40-60 bp ends homologous to the target genomic locus. The homologous ends are provided in the PCR primers, whose 3′ ends are homologous to the ends of the cassette used to delete or tag any target gene. This protocol described the default PCR conditions that are compatible with most deletion or tagging cassettes.

Materials:

plasmid containing desired cassette

Expand LT Buffer 3

Expand Long Template PCR enzyme

dNTP mix 2.5 mM dil.

water, nuclease free

Reaction volumes:

1 ul template plasmid cassette (100 ng)

5 ul Expand buffer 3

4 ul dNTPs (2.5 mM)

1 ul forward primer (2.5 uM)

1 ul reverse primer (2.5 uM)

0.5 ul Expand LT Polymerase

37.5 ul nuclease free water

50 uL Total Volume

PCR Protocol:

SimpliAmp Thermocycler

1) 2 min 95 °C, 2) 30 s 95 °C, 30 s 55 °C, 3 min 69 °C, repeat 2 35x, 3) 5 min 69 °C, 4) hold 4 °C

If mis-priming is a problem (i.e. smearing or bands smaller or larger than desired band) use the following:

1) 2 min 95 °C, 2) 30 s 95 °C, 30 s 60 °C, 3 min 69 °C, repeat 2 5x, 3) 30 s 95 °C, 30 s 55 °C, 3 min 69 °C, repeat 3 30x, 4) 5 min 69 °C, 5) hold 4 °C

If low product yield is a problem (i.e. very faint band of correct size) use the following:

1) 2 min 95 °C, 2) 30 s 95 °C, 30 s 45 °C, 3 min 69 °C, repeat 2 5x, 3) 30 s 95 °C, 30 s 55 °C, 3 min 69 °C, repeat 3 30x, 4) 5 min 69 °C, 5) hold 4 °C

Perform  Agarose gel electrophoresis of DNA  as described.

 Confirming genome edits

Yeast gene deletions or gene fusions (e.g. ORF with epitope tags or FP, promoter replacement, MS2 mRNA tagging) can be generated simply by transformation into yeast of a PCR amplicon containing the desired selectable marker or tag flanked by 40-60 bp ends homologous to the target genomic locus. After obtaining transformants carrying the selectable marker, it is necessary to test four (usually) transformed colonies for integration of the marker cassette into the correct genomic location. This can be done by performing PCR reactions using genomic DNA (Yeast DNA Isolation), and test primers designed as described in the Gene Deletion Primer Design, or the Gene Tagging Design protocols. This protocol describes the default PCR conditions that are compatible with test PCRs on isolated gDNA.

Materials:

plasmid containing desired cassette

Taq DNA Polymerase, recombinant NEB

standard Taq DNA Polymerase buffer

dNTP mix 2.5 mM dil.

water, nuclease free

yeast genomic DNA

forward test primer

reverse test primer

Reaction volumes:

1 ul gDNA

2 ul Taq buffer

2 ul dNTPs (2.5 mM)

1 ul forward test primer (2.5 uM)

1 ul reverse test primer (2.5 uM)

0.2 ul Taq polymerase

12.5 ul nuclease free water

20 uL Total Volume

PCR Protocol:

SimpliAmp Thermocycler

If performing PCR for gene deletions use the following:

1) 2 min 95 °C, 2) 30 s 95 °C; 30 s 60 °C; 1 min 72 °C; repeat 5x, 3) 30 s 95 °C; 30 s 55 °C; 1 min 72 °C; repeat 35x, 4) 5 min 69 °C, 5) hold 4 °C

If performing PCR for tags use the following:

1) 2 min 95 °C, 2) 30 s 95 °C, 30 s 60 °C, 3 min 69 °C, repeat 2 5x, 3) 30 s 95 °C, 30 s 55 °C, 3 min 69 °C, repeat 3 30x, 4) 5 min 69 °C, 5) hold 4 °C
Perform  Agarose gel electrophoresis of DNA  as described.

 

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