Quick Yeast DNA Prep: Isolation of Total DNA (genomic and plasmid)
来自 : 蚂蚁淘
QuickYeastDNAPrep:IsolationofTotalDNA(genomicandplasmid)
LindaHoskins,HahnLab10/16/98
- Growa5mlYPDO/Ncultureinoculatedwithasingleyeastcolonyat30deg.
- Transferculturetoasmall13x100glasstube.Spindowncells2min.intabletopcentrifuge.Pouroffsupernatant.
- Washcellswith5mlH2O.Spindowncells2min.Pouroffsupernatant.Cellpelletcanbestoredat-20deg.
- ResUSPendcellsin500ullysisbufferbyvortexing.
Lysisbuffer: | | 20ml: |
0.1MTrispH8.0 | | 2ml1MTris8.0 |
50mMEDTA | | 2ml0.5MEDTA |
1%SDS | | 2ml10%SDS |
| | 14mlH2O |
| | |
Addacid-washedglassbeads(400-500microns)toabout2mmbelowmeniscus.Vortex30sec.Add25ul5MNaCl.Vortex30sec.Spindown2min.todecreasefoam.RemovelysedcellswithP1000atbottomoftubeandtransfertoa1.5mlmicrofugetube.Add400ulofTE-saturatedphenol.Vortex.Spin4min.Transferupperphasetoaclean1.5mlmicrofugetube(~400ul).Add400ulphenol:chloroform(4:1).Vortex.Spin4min.Transferupperphasetoaclean1.5mlmicrofugetube.Add1ml95%EtOHandmix.Spin6min.PouroffEtOH.Washwith1ml70%EtOH.Vortex.Spin6min.PouroffEtOH.Drypelletinhoodorvacuum.Resuspendin50ulTE(vortex,37deg.10min,vortex)
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