2011030014宋予晴

Title:

GeneChipExperiment

Background:

Objectives:

Learnabouttheknowledgeoftheprinciplesandmethodsofgenechipexperiment

Usegenechiptechnologytoidentifythebacteriawhichincludeschiphybridization,washing,scanninganddataprocessing

Principle:

Genesequencesfromvariousbacteriawereprintedonthemicroarrayslides

AmplificationoftheinterestingsequencesbyPCR

PCRproductslabeledwithfluorescentmolecules

HybridizationwiththelabeledPCRproducts

Chipwashing

Chipscanninganddataanalysiswiththemicroarrayscannerandsoftware

Identifythetypeofgenesbeinghybridized

DeducethetypeofbacteriaexistedinthePCRamplifiedtemplate

Materials&Equipment:

Bacteriatobeidentified:Escherichiacoli,Xanthomonascampestris

Genechip:TestchipforbacteriaidentificationfromNERCBBT

PCRproducts(labeledwithfluorescentmolecules)

Instruments:晶芯?LuxScan-100microarrayscanner,waterbath,shaker

Procedure:

ChipHybridization

Add200μlofwaterinthegrooveonthebottomofthecassette.

Placethechip(gridfacingup)inthebottomofthecassette.

Usepipettetoadd12μldifferenthybridizationsolutionintotheostioleonthecoverslip

Placethecoverslipsontopofthechip.Makesurethatthesidewiththeprotrudingfeaturesfacesdown.

Putthechipinto42℃waterbathfor1.5hour.

Thehybridizationsolution

Initialconcentration

Finalconcentration

Sampleamount

100％formylamine

50％formylamine

6μl

10％SDS

0.2％SDS

0.24μl

20×SSC

3×SSC

1.8μl

PCRproduct

4μl

ThefirstistheE.coli,thesecondistheXanthomonascampestris,thethirdis2μlofE.coliandXanthomonascampestriseach,thelastoneisthecontrol

ChipWashing

WashingbufferI:2×SSC,0.2%SDS

WashingbufferII:0.2×SSC

Afterhybridization,quicklymovethechipintothewashingboxwithgridfacingup,usewashingbufferItowash4minutes.

Quicklyremovethechipintoanotherbox,usewashingbufferIItowash4minutes.

Scanning

Scantheresult

Result&Analysis:

OnlyE.coli

OnlyXanthomonascampestris

BothE.coliandXanthomonascampestris

Controlgroup(neitherE.coliorXanthomonascampestris)

Thethreelineswithwhitesquareframearepositivecontrols,thelinewithredsquareframeissignalsfromE.coli,andthelinewithbluesquareframeissignalsfromXanthomonascampestris.

Therewassomepollutionin