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CHO细胞染色体制备[Harvard Medical School]

MitshisonLab,DepartmentofSystemsBIOLOGy,HarvardMedicalSchoolhttp://mitchison.med.harvard.edu/protocols/chr1.html

Buffers:

SwellingBuffer(PME):

5mMPIPES,pH7.2
5mMNaCl
5mMMgCl2
1mMEGTA

Recipe:

1.25ml0.2MPipespH7.2
50ul5MNaCl
250ul1MMgCl2
0.625ml0.4MEGTA
47.8mlwater
storeat30degC

LysisBuffer:PMED(PME0.1%digitonin)5ug/mlLPC.Storeonice;addLPCanddigitoninjustbeforeuse.

SucrosestepgrADIents:30%,40%,50%and60%inPME5ug/mlLPC

30%:
2.2ml2Msucrose
2.3mlwater
0.5ml10XPME
40%:
2.9ml2Msucrose
1.6mlwater
0.5ml10XPME
50%:
3.6ml2Msucrose
0.9mlwater
0.5ml10XPME
60%:
4.4ml2Msucrose
0.1mlwater
0.5ml10XPME

storeonice;addLPCjustbeforepouringgradient(seebelow)

Otheritemsrequired:

  1. Chilled15mldouncewithtightpestle(BforKontes;AforWheaton)
  2. 215mlCorextubesonice
  3. HB-4rotorat4CandclinicalatRT

Protocol:

  1. ArrestCHOcells(~70%confluent)using8-10ug/mlvinblastinefor8-10hours
  2. Makeallbuffersandstoreatappropriatetemperaturebeforestartingprep
  3. Collectmitoticsbyblowoff
  4. Spininclinicalat#5for3"tocollectcells
  5. SwellcellsbyresUSPendingin10mlSwellingbuffer(30deg.C),addingadditional40mlswellingbufferandincubatingat30deg.Cfor5".
  6. Pelletswollencellsinclinicalat#5for3".Duringsteps4&5dothefollowing:
    1. adddigitonintolysisbufferandLPCtolysisbufferandsucrosegradientsolutions
    2. pourstepgradient:
      • 3mls60%
      • 2mls50%
      • 2mls40%
      • 2mls30%
    3. Ensurethatdounceisicecoldwithpestlein.
  7. CRITICALSTEP
    1. Aspiratesupeandvigorouslypipet7mlsofice-coldlysisbufferontocellpellet.
    2. Rapidlypipetupanddownonceandpipetthecellsuspensionintothedounce
    3. Snapthepestleupimmediatelyandquicklyapply3-4strokesbeingcarefulnotto"pop".Thisiscriticaltoincreaseyieldofsinglechromosomes;delayatthisstepwillcausethechromosomestocluster
    4. Apply15strokestotalwaiting~2secsbetweeneachstrokebeforelookingatthelysatebymixingwithalittleHoechst
  8. Transferlysatetoa15mlconicalonice
  9. SpininHB-4for1"at900rpm(topelletnuclei)at4deg.C
  10. Layersupeontosucrosestepgradientandspin5K15"4deg.Cwithbrakeoff.
  11. Aspiratetillthemiddleofthe40%stepandcollectflocculentwhitematerialatthe40-50%and50-60%interfacesusingapasteurpipet.
  12. Mixwelltoresuspendthechromosomesandaliquotinto10ulaliquots.Store-80


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