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SETTING UP A PCR LABORATORYSETTING UP A PCR LABORATORY
ByBy
Prof. Dr.Prof. Dr.AsmaaAsmaaHusseinHussein
Prof. ofProf. ofZoonosesZoonoses& Director of the&
Director of the
MBRUMBRU
Assiut UniversityAssiut University
The PCRThe PCRlaboratory should consist of threedistinct work
areas. In order to avoid thecontamination problems, each area
should bededicated to a single procedure.
Specimen preparation occurs in the first
area, reagent preparation and PCR set-up inthe second area, and
amplification anddetection in the third area.
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SAMPLESAMPLE
PREPARATIONPREPARATION
REAGENTREAGENT
PREPARATIONPREPARATION
& PCR SET& PCR SET--UPUP
AMPLIFICATION AND DETECTIONAMPLIFICATION AND DETECTION
Positive-displacement pipettes or pipettorswith
aerosol-resistant tips.
Gloves & laboratory coat.
Refrigerator, freezer, water bath or dry
heat block laminar flow biosafety cabinet.
Cell lysis reagents.
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Amplification reagents & supplies.
Positive-displacement pipettes or pipettorswith
aerosol-resistant tips.
Laminar-flow biosafety cabinet or dead airbox.
Gloves & laboratory coat.
Refrigerator & freezer.Water bath or dry heat block.
Thermal cycler.
Pipettors with aerosol-resistant tips.
Detection equipment (electrophoresis unit,incubator, plate
washer, plate reader, waterbath).
Refrigerator & freezer.
Reagents and supplies for detection.
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The following practices will diminishThe following practices
will diminish
the potential for contamination:the potential for
contamination:
Each area should have dedicated supplies andreagents.
Color coding of reagents and supplies identifiesthose that
belong to a particular area.
Reagents, supplies and equipment should neverbe taken from one
area to another, three sets of
pipettors are therefore essential.
The workflow must be unidirectional fromclean (pre-PCR) to dirty
(post-PCR).
Dedicated labcoats and gloves should be worn at
each work site; when moving to a new area,workers should put on
new gloves and labcoats.
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Flow of samples forFlow of samples forPCR analysisPCR
analysis
PrePre--PCRPCRis the protocols and equipmentrequired for the
isolation of nucleic acid and the
assembly of the reaction to amplify the samples
Bench spaceBench spaceDoorDoor
TissueTissuecultureculture
incubatorsincubators
MicrobiolMicrobiol..SafetySafetyhoodhood
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UprightUprightFrzrFrzr
44 CC Frig.Frig.
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SinkSink
Type 1Type 1HH22OO
UV/VISUV/VISSpectroSpectro..
CentrifugeCentrifuge
Slight PositiveSlight PositiveAir PressureAir Pressure
PrePre--PCR Lab.PCR Lab.
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Bench spaceBench spaceDoorDoor
PostPost--PCR Lab.PCR Lab.
Slight NegativeSlight NegativeAir PressureAir Pressure
PCPCw/networkw/networkconnectionconnection
ThermalThermalcyclerscyclers
ThermalThermalcyclerscyclers
GelGelelectrophoresiselectrophoresis
equipmentequipmentHybHybovenoven
RealReal timetimePCR instrumentPCR instrument
44 CC Frig.Frig.
--2020 CCUprightUpright
FrzrFrzr
SinkSink
GelGelimagingimagingsystemsystem
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Strict adherence to proper laboratoryStrict adherence to proper
laboratory
technique:technique:
Physically isolate PCR preparations and productsPhysically
isolate PCR preparations and products
Autoclave solution.Autoclave solution.
Aliquot reagents.Aliquot reagents.
Use disposable gloves and change gloves oftenUse disposable
gloves and change gloves oftenduring setduring set--upup
Avoid splashes.Avoid splashes.
PremixPremix reagents.reagents.
Add DNA last.Add DNA last.
Choose positive and negative controls carefully.Choose positive
and negative controls carefully.
Use positiveUse positive--displacement pipettes or
aerosoldisplacement pipettes or aerosolresistant tips on
airresistant tips on air--displacement pipettesdisplacement
pipettes
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Thank youThank youforforyouryour
attention!attention!
