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  • Yeast Transformation: Supplies and Reagents

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    Cecelia Dot Dot
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    This document provides a protocol for transforming yeast cells. It lists the necessary supplies which include DNA, yeast strain, growth media, and reagents for the transformation mix. The procedure describes growing yeast overnight, preparing cells by centrifugation, adding the transformation mix containing DNA, incubating at 30°C and 42°C, and plating on selective media to isolate transformed colonies. Controls without DNA are also plated to check for background growth.

    Original Description:

    yeast molecular biology

    Original Title

    Yeast Transformation

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    This document provides a protocol for transforming yeast cells. It lists the necessary supplies which include DNA, yeast strain, growth media, and reagents for the transformation mix. The procedure describes growing yeast overnight, preparing cells by centrifugation, adding the transformation mix containing DNA, incubating at 30°C and 42°C, and plating on selective media to isolate transformed colonies. Controls without DNA are also plated to check for background growth.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PDF, TXT or read online from Scribd
    Flag for inappropriate content
    13 views2 pages

    Yeast Transformation: Supplies and Reagents

    Uploaded by

    Cecelia Dot Dot
    This document provides a protocol for transforming yeast cells. It lists the necessary supplies which include DNA, yeast strain, growth media, and reagents for the transformation mix. The procedure describes growing yeast overnight, preparing cells by centrifugation, adding the transformation mix containing DNA, incubating at 30°C and 42°C, and plating on selective media to isolate transformed colonies. Controls without DNA are also plated to check for background growth.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PDF, TXT or read online from Scribd
    Flag for inappropriate content
    of 2

    January2010

    YeastTransformation

    SuppliesandReagents
    1.
    2.
    3.
    4.
    5.
    6.

    DNA(cutplasmidorpurifiedPCRproduct)
    Appropriateselectionplates

    YeastStrain

    SterileYPD
    100mLLiAc
    TMix:
    I.
    50%PEG
    II.
    1.0MLiAc
    III.
    10mg/mLSSDNA(denaturedbyheatingto95Cfor10minandimmediately
    chillingonice;usablefor4freeze/thawcycles).
    7. SterileH2O
    8. Microfugeat7500rpm(60008000rpm)
    9. Centrifugeat3000rpm
    10. 30Cheatingplate
    11. 42Cwaterbath
    Day1:Growyeast
    1. Growyeaststrain(s)________________overnightat30CinYPD.
    (1yeastcolonytobetransformedin5mLYPD)
    Day2:Transformyeast
    1. Inoculate50mLofsterileYPDwith700LofYPDovernightculturein250mLflask.
    2. Growinshakingincubatorfor35hrsat30C.
    3. Harvestcellsbycentrifuging(Eppendorfcentrifugemodel5702)at3000rpmfor1min.
    Removesupernatant.Resuspendpelletin25mLsterileH2O.
    4. Pelletagain.Removesupernatant.Resuspendpelletin1mLof100mMLiAc.
    5. Transferto1.5mLmicrofugetube.Centrifugeat3000rpmfor1min,remove
    supernatant.Resuspendcellsin400Lof100mMLiAc.
    6. Aliquot50Lofcellsuspensioninto1.5mLtubes(1foreachtransformation).Pelletcells
    andremovesupernatant.
    7. Addsequentiallytoeachtube300LofTransformationmixaccordingtotable:

    M CleanLabProtocol

    1of2

    January2010

    Order Reagent
    1
    50%PEG3350
    1.0MLiAc
    2
    3
    ssDNA(10mg/mL)
    o
    4
    sterileH2O
    5
    DNA

    Volume/Conc.
    240L
    35L
    25L
    50L
    2040L(.110g/L)

    8. Vortextoresuspendcells.
    9. Incubatefor30minat30C.
    10. Incubateinwaterbathat42Cfor2025min(upto40min).
    11. Microfugeat60008000rpmfor1min.Removesupernanttransformationmixwith
    pipet.
    12. Add1mLofYPDandincubatefor1hrat30C(orovernightforhighertransformation
    efficiency).Thisstepisonlynecessaryfortransformationswithdrugmarkers.For
    transformationswithnutritionalmarkerscontinueimmediatelytothenextstep.
    13. Pelletandremovesupernatant.Resuspendcellsin200Lofsterilewaterbypipettingup
    anddowngently.
    14. Platecellsuspensiononantibioticselectionplates:

    Plate# Yeastcellsuspension sterileH2O


    1
    20L
    160L
    2
    180L
    0L

    15. Includepositiveandnegativecontrols:
    Positivecontrol:100Lyeastcellsuspension(noplasmid)+100LsterileH2Oon
    YPDplate.
    Negativecontrol:100Lyeastcellsuspension(noplasmid)+100LsterileH2O
    onappropriateselectionplate.
    16. Incubateplatesat30C(coloniesshouldappearin34days).

    M CleanLabProtocol

    2of2

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