DNAExtractionMethods

Phenol-chloroformDNAextractionfromsandflies

1.CrashIndividualsandfliesin0.5mllysisbuffer(50mMNaCl,10mMEDTA,50mMTris-HClpH8).2.IncubateThesandflyhomogenateswith100ng/mlRnaseat37؛Cfor30minutes.

3.IncubateThecelllysateswith200ng/mlProteinaseKat65؛Cfor2hours.

4.ExtracttheDNAwithequalvolumesofbufferedphenol,Phenol-chloroform-isoamylalcohol(v/v,25:24:1)

andfinallychloroform-isoamylalcohol(v/v,24:1).

5.precipitatetheDNAwith3-Mammoniumacetateand2.5volumeof100%ethylalcohol.

6.washtheDNApelletwith70%ethanol

7.drythepelleteinspeedvacuumcentrifugefor10minutes.

8.sUSPendtheDNApelletswith100-µldoubldisteled,sterilewaterandstoreitat-20forPCRexperiments.

Guanidinethiocyanateextractionprocedure

Reagentspreparation

1.Guanidinethiocyanate(FLUKA,Switzerland)solutioncontains4-Mguanidinethiocyanate,0.1MTris-HClpH6.4,0.02MEDTApH8and1.3%TritonX-100.

2.Sodiumiodide(Merck,Germany)ispreparedasa6-Msolution.

3.SuspendThreegramsofsilicabeads(Sigma)in25mlofdoubledistilled,sterilewaterfor24hours.

4.CentrifugeThesuspensionat12,000RPMfor5secondsandremovethesupernatant.

5.Addadditional25-mlofdoubledistilled,sterilewaterandwashthebeadsfor5hoursusingrotator.

6.centrifugeThesuspensionat12,000RPMfor5secondsandremovethewater.

7.Finally,add30µlof1Mofhydrochloricacid(HCl)andautoclavethebeads

8.aliquotandstoreinthedarkatroomtemperature.

Washingbuffer

Thewashingbufferstockcontained;0.2MTris–HClpH7.5,1Msodiumchloride,and20mMEDTApH8.1.Dilutethewashingbufferstocksolution1:9withdouble,distilled,sterilewater

2.Addanequalvolumeof100%ethylalcohol.

3.Dividethesolutioninto50-mltubesandstoreat–20؛C.

Procedure

1.groundEachsandflyin1.5mlautoclavedandUVrADIatedmicrofugetubes,usingamicro-pestle

(Ependorph,Germany).

2.suspendeachpestlein0.5ml4-Mguanidinesolutionandincubatethesamplesat56؛Cundergentle

agitation.

3.Nextday,boilthesamplesfor10minuetsandcentrifugethemat12,000RPMfor5minutes

4.Removethedebrisleavingasandflytissuepellet.

5.Add1mlof6Msodiumiodideand10µlofsuspendedsilicabeadstoeachtube,vortexgentlyfor5

secondsandincubateonicefor1hour,performgentlemixingevery15minutes.

6.Removethesupernatantcarefullyandwashthepellettwotimeswith500µlofice-coldwashingbuffer.

7.vortexthepelletandcentrifugedfor5secondsat12,000RPM.

8.Removethebufferandwashthepelletonetotwotimeswith100%ethylalcohol.

9.Removeethylalcoholanddrythepellet.

10.ResuspendtheDNAin100µlofdoubledistilled,sterilewater,mixgentlyandincubatedat56؛Cfor1

hour.

11.CentrifugetheDNAsamplesat12,000RPMfor5minutesatroomtemperature,aliquotthesupernatant

andstoreat–20؛CforPCRexperiments.