Fungal Genomic DNA Extraction 专区
来自 : 蚂蚁淘
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| Author:Dr.EricW.Boehm |
| Source:ContributedbyDr.EricW.Boehm |
| Abstract:Thisproceduredoesnotrequirephenolextraction.TheDNAispureenoughforrestrictiondigests,PCRandgenomiclibraryconstruction. |
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Overview Highthroughputofmanyfungalisolatescanbeachievedbygrowingaxenicculturesineither(a)1.5mLmicrofugetubes,halffullwithliquidmedia(500uL),withaholepunchedthroughthetopforaeration;orifgreatervolumesarerequired,(b)peteriplatescontaining10mLliquidmedia.Inbothcases,mediaisdecantedandseveralwaterwashesdonetoremovemediacarbohydrates. Procedure - Ifusingpetriplates,the3-7daycultureisthendicedup,usingasterilescalpel,andsmallpieces(ca.50-100mg)areplacedinmicrofugetubescontainingsterilesand(ca.100mg)and500uLofextractionbuffer.Ifusingmicrofugetubes,simplyaddthesandandextractionbuffer:
- 100mMTris,pH8.0
- 10mMEDTA
- 2%SDS
- 100ug/mLProteinaseK
- 1%B-mercaptoethanol
(tubescanbestoredinthisextractionbufferatminus20Cforgreaterthanayear) - UsingaKontesmicro-homogenizerwithsterilizedtips(FisherScientificCat.#K749540-0000)samplesaregroundintoaslurryandincubatedfor60min.at60C.
- Saltconcentrationisadjustedto1.4Mwith5MNaCl,1/10vol.of10%CTABaddedandsamplesincubatedafurther10min.at65C.
- Add1vol.chloroform:isoamylalcohol,gentlyemulsifybyinversion,incubateat0Cfor30min.Spin10min.at4Catrpmmax.Transfertopphasetofresh1.5mLmicrofugetube,add1/2vol.5MNH4OAc,mixgently,icefor60min.;spinat4Catrpmmax.
- Transfersupernatanttofreshtube(addstockRNase10mg/mLtoafinalconcentrationof0.02ug/uL)add0.55vol.isopropanoltoprecipitatetheDNA.Spinimmediately5-10min.atrpmmax.Aspirateoffsupernatant,washDNApellettwicewith70%ETOH,airdrypellet20min.andresUSPendin50uLTEbuffer.Incubate4Covernight.
- QuantifyyieldwiththeHoeferDyNAQuant200fluorometer.
Thisproceduredoesnotrequirephenolextraction.TheB-mercaptoethanolcanbeomittedfromtheextractionbufferforsafety,butyieldswillbeslightlylower.TheDNAispureenoughforrestrictiondigests,PCRandgenomiclibraryconstruction. Note ModifiedfromMoller,Bahnweg,SandermannandGeiger,1992,NucleicAcidsRes.20:6115-16. |
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