Preparation of metaphase slides for molecular cytogenetic experiments

Criteria of good slides for molecular cytogenetic experiments Longer chromosome Well spreading : without too much overlapping chromosomes Little cytoplasmic backgrounds

Longer chromosomes Synchronized culture –Methotrexate (MTX) blocking method –Thymidin methods –EtBr methods

Chromosome spreading Factors influencing the spreading –Relative humidity –Temperature –Drying time of the slides –Distance of dropping cell onto slide

Dynamics of chromosome spreading Time ab c d Fixative layer Spurbeck et al.,1996

Basel et al.,1997

Main factor –drying time of fixative solution (methanol, acetic acid) –Relative humidity –Temperature

Thermotron CDS-5 cytogenetic drying chamber

How we do? Prepare slide in a good weather Slides should be very clean Using dry slide Before or after dropping cell, add one or two drop fixative solution on slides

More fixative or acetic acid

A new method

Storage of slides Short term : 4 o C in EtOH Long term : -20 o C –in EtOH or –in a box filled with N 2 gas

Aging of slides Oven aging for G banding Room temperature aging –about two weeks for CGH –Few days for FISH Chemical aging

2X SSC at 37 o C for 30~60 min EtOH aging RT  94 o C for 2-20s  RT (1-2 o C/s)

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