国际化学品安全卡(中文版)
来自 : 蚂蚁淘
RapidMethodforPreparationofGenomicDNAfromP.aeruginosa- Inoculate1mlofL-brothorotherrichmediawithcellsofthestrainfromwhichgenomicDNAistobeisolated.Growthiscultureat37°Covernightonaroller.
- Transferthisovernightculturetoa1.7mlmicrofugetube.Pelletthecellsbycentrifugationonhighatroomtemperatureforoneminute.
- ResUSPendandwashthecellsin1mlofTNEbufferbypipettinggentlyupanddownuntilthepelletiscompletelyresuspended.
- Pelletthecellsbycentrifugingasbefore.
- Resuspendthepelletin135µlTNEbuffer.
- Add135µlofTNEbuffercontaining2%TritonX-100.
- Add30µlfreshlypreparedlysozyme(5mg/ml).Mixwellbytappingthetube.
- Incubateina37°Cwaterbathfor30minutes.
- Add15µlproteinaseKsolution(20mg/ml).Mixwellbyinvertingthetubeseveraltimes.
- Incubateina65°Cwaterbathfor2hours.
- GenomicDNAthuspreparedmaybekeptat-20°Cuntilused.
- DigestionoftheDNAisbestaccomplishedbyallowingthedigestiontoproceedovernight,andbykeepingthevolumeofDNAtolessthanone-fifththetotalvolumeofthedigestionmixture.ThereissufficientDNAineach50µldigestionforthreeelectrophoreticrunsona14.5x20cmgelsuitableforSouthernblotting.
DNA preparation 8.4 µl TNEBufferdH2O34.6µl10Xenzymebuffer5.0µlTris-HCl,pH8.010mMRestrictionenzyme(20-30U)2.0µlNaCl10mMTotalvolume:50.0µlEDTA,pH8.010mM
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