Preparation of singlestranded probes from cloned cDNA

a)2-5µgofplasmidDNAcontainingtheCDNAinsertislinearizedusinganappropriaterestrictionenzyme.Forantisenseprobes,auniquerestrictionsite5"totheinsertisused.ThisdigestedDNAwillbeamplifiedusinganantisenseprimeratthe3"endoftheinsert.Forsense(control)probes,auniquerestrictionsite3"totheinsertisused.ThisdigestedDNAwillbeamplifiedusingasenseprimeratthe5"endoftheinsert.(Insertsofupto2kbarelabeledefficiently).

b)Add4XSTOPtodigestedDNAtoafinalconcentrationof1XSTOP.

Extractoncewithphenol/chloroform,oncewithchloroform,andprecipitatewith3volumesof100%EtOH.

ResUSPendinTEatafinalconcentrationof100-200µg/ml.(checkconcentrationonagel).

c)Mixthefollowingreagentsin0.5mlEppendorftube.

water7.0µl

10xTaqBuffer2.5µl

25mMMgCl21.5µl

10xDIG-labeleddNTPmix5.0µl

Primer*(30ng/µl)5.0µl

DigestedDNA(100-200µg/ml)2.0µl

mineraloil40µl

*ForcDNAsclonedinBluscript,weusethefollowingprimers(21mers):

"T3long"=5"-ACTAAAGGGAACAAAAGCTGG-3"

"T7long"=5"-ACTCACTATAGGGCGAATTGG-3"

d.Boilreagentsfor5min,placeonice,beforeadding2.0µlofa1:8dilutioninwaterof5units/µlTaqpolymerasestock(1.25unitstotal).

e.Incubatelabellingreactionfor35thermalcyclesasfollows:

95°Cfor45seconds

55°Cfor30seconds(lowertemperatureforprimerslessthan20nt)

72°Cfor1minute

f.75µlofH20isaddedtothereactionbelowtheoiland90-95µlofthedilutedreactionistransferredtoanewtube.

g.10µlof1MNaCl,10µgofglycogen,and3volsof100%EtOHareaddedtothedilutedreaction.After30minat-70°C,thereactioniscentrifugedat15,000rpmfor10min.Thepelletiswashedin70%ethanol,dried,andresuspendedin300µlofhybridizationbuffer.

h.Theprobeisboiledfor1-2hours.Thisstepreducesthelengthoftheprobeforefficientpenetrationofembryos.

e.Probeproductionisassayedusingthefollowingprotocol.1µlofprobeinhybridizationbufferismixedwith5µlof5xSSC,boiledfor5min,andcooledonice.1µlofthismixtureisspottedonanitrocellulosestrip.Severaldilutionsofapre-labelledcontrolDNA(1ngto1pg/µl;BoeringherMannheim)arealsospottedforcomparison.Thestripisbakedfor30mininavacuumovenat80oC,washedoncein2xSSC,twiceinPBT,andblockedfor30minutesinPBT.Thestripisthenincubatedfor30-60minwithAP-anti-DIGantibodydiluted1:2000inPBT.Afterthree10minwashesinPBT,andtwo5minwashesinstainingsolution,thestripisdevelopedinstainingsolutioncontaining4.5µlNBT/ml,3.5µlX-phosphate/ml.SpotsshouldbevisIBLewithinminutes.Spotintensitiesoftheprobeandcontroldilutionsarecomparedtodeterminetheconcentrationoftheprobe.

f.Probescanbestoredat-20oCinhybridizationbufferforseveralweeks.