YeastTransformationUsingFrozenCompetentCellsandSingle-strandedDNAasaCarrier

KatherineKolor

SchiestlandGietz(1989)Curr.Genetics16:339-346

Gietz,etal(1995)Yeast11:355-360

Preparingcompetentcells:

1.Inoculate100mlyeastsyntheticcompletemedium+3%glycerolmediumfromovernightculture.Growcultureto0.5-1.0X10⁷cells/ml.(2-3Xhigherefficiencyifdilutedbackto2X10⁶cells/ml,thengrown2-3moredoublings.)Note:Thecellsmustbegrowninglycerolifyouaregoingtofreezethem.Glucose-growncellslosecompetenceuponfreezing.

2.Pelletcellsgently(~800xg).ResUSPendin7-8mlof1XTE-LiAc.

3.Pelletcellsgently.Resuspendin1mlof1XTE-LiAc.

4.Rotateat23^oCfor1.0-1.5hr.

CellscanbefrozenatthispointforfutureusebywrappingthetubeinseverallayersofKimwipesand/orpapertowelsandplacingtheminanicecreamcontainerina-70^oCfreezer.Itisimportanttowrapupthetubesothatthecellsfreezeslowly.Thawcellsatroomtemperaturefortransformation.

Transformation:

1.Boil20microlitersofherringspermDNA(8mg/ml)inanEppendorftubefor15min.Coolonice5min.

2.AddtransformingDNA(upto5micrograms)tothecarrier.

3.Add100microlitersofcompetentcellstotheDNA.

4.Add700microlitersofPEG-TE-LiAcsolution(madefreshfromthestocksolutions).Vortexbrieflyandgentlytomix.

5.Rotatetubeat30^oCfor30min.

6.Transfertubeto42^oCwaterbath,incubatefor15min.

7.Plateanaliquotoftransformationmixdirectlyontoselectivemedium.

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Stocksolutions

50%Polyethyleneglycol4000

1MTris-HClpH8.0

1MLithiumacetatepH7.5

0.2MEDTApH8.0

1XTE-LiAc

10mMTris-HClpH8.0

1mMEDTA

0.1MLithiumacetate

PEG-TE-LiAc

40%PEG-4000

10mMTris-HClpH8.0

1mMEDTA

0.1MLithiumactetate

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