INJECTIONANDHOLDINGPipetteS

Theglasscapillarytubingusedshouldbethinwalled,borosilicateglasswithoutafibre.

e.g.ClarkElectromedicalInstruments

GC100T-15(=15cm)

GC100T-10(=10cm)

OUTSIDEDIAMETER:1.0mm

INSIDEDIAMETER:0.78mm

LENGTH:150mm

AustralianAgents:

SDRClinicalTechnology,213EasternValleyWay,MiddleCoveNSW2068(02)9582688

Glassshouldbeacidwashed:-

1.Placeglassina100mlmeasuringcylinder,containing1:1v/vSulphuricacid,Nitricacid,soakovernight.Thisisdoneinfumehood,makesureglovesandgogglesareworn.

2.Pouracidintoanothercylinder,thiscanbereused.

3.Washglassatleast10xwithMilliQH20then3xwithTravenolH20.Drainwell.

4.CovercylinderwithParafilm,pokeholesinitusinganeedleandplaceglassin60¡Covenuntildry.Thistakesafewdays.

Pipettepulling:-

FlammingBrownmicropipettepullerModelP-87

SutterInstrumentCo.USA

UsingProgram6(VH)

Heat=915Pull=20Vel=90Time=120Pressure=500

Injectionpipettes:-

Injectionpipettesshouldhaveaninternaldiameteronlymarginallylargerthanthestemcells(18-20um)andasharppointwhichallowstheneedletopenetratecleanlyintheblastocoelcavity.

Theglassiscutbyhandunderastereodissectingmicroscope,atamagnificationofX50,usingasharpscalpelbladeandalargeglasspetridishfilledwithsilicon.(Dow-corningSilgard).Theglasswillusuallybreakwithasharpbevelledendwhentouchedwiththeedgeofthescalpelblade.Theresultantpipetteshouldhavearelativelylongsectionattheappropriatedimater(18-20um).

Holdingpipettes

Holdingpipettesarebluntflame-polishedpipettesthroughwhichsuctionisappliedtoimmobilizetheblastocyst.Thesamepieceofglassiscut,onlyfurtheruptheshaft~200um.Thisiscutbyholdingtheshaftofglassonyourforefingerwithyourthumbandscoredwithadiamondpencilthenbrokenbyflickingthesurplusoffthetop.Acleanneatbreakisessentialotherwisetheblastocystdoesnotsitproperlyontheendandcomesoffduringtheinjectionprocess.

Theendofthepipetteisthenflamepolishedbyholdingitinamicroburnerflameuntiltheinternaldiameterisreducedto20um.(1-2divisions).