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HIV Drug Resistance Training. Module 4: Molecular Lab Set-up and Workflow. Topics. Principles of Lab Set Up Other Workflow Considerations Application of Principles. Objectives. Identify principles for laboratory setup and workflow design that prevents lab contamination.

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Hiv drug resistance training

HIV Drug Resistance Training

Module 4:

Molecular Lab Set-up and Workflow


Topics

Topics

  • Principles of Lab Set Up

  • Other Workflow Considerations

  • Application of Principles


Objectives

Objectives

  • Identify principles for laboratory setup and workflow design that prevents lab contamination.

  • Identify examples of good/poor practices in lab setup and workflow.

  • Given the current lab setup, identify changes that must be made to prevent lab contamination.


Principles of lab set up

principles of lab set up

What is the ideal set up for a molecular lab?

What compromises can—and cannot—be made when the ideal is not possible?


Amplification of pr rt sequences

Amplification of PR/RT Sequences

Reagent Preparation

SpecimenPreparation

Sequencing

Amplification

0

1012

1010

105

Copy #


Genotyping assay procedures

Positive and negative controls

Positive control

Genotyping Assay Procedures

Patient virus

RNA Extraction

cDNA synthesis (RT)

cDNA amplification (PCR)

Sequencing reactions

Electrophoresis

Data Analysis (base calling)

Report (sequence, mutations, interpretation)


Ideal lab workflow for genotyping nested pcr in house assays only

Ideal Lab Workflow for Genotyping(Nested PCR/In-House Assays Only)

SPECIMENS

Frozen plasma

Whole blood

DBS

LAB 2

"Specimen Processing" Lab

Specimen receipt & storage

RNA extraction and 1st round PCR set-up

LAB 4

"Post-amplification" Lab

2nd round amplification

Gel electrophoresis

Sequencing reactions

Capillary electrophoresis

LAB 1

"No Template" Lab

Reagent storage and

Master mix preparation

LAB 3

“1st Round PCR” Lab

1st round amplification

2nd round PCR set-up


Example of a four lab layout nested pcr in house assays only

-20°C

freezer

-20°C

freezer

-20°C

freezer

-80°C

freezer

-80°C

freezer

-20°C

freezer

BSC

BSC

Example of a Four-Lab Layout (Nested PCR/In-House Assays Only)

Ice machine

sink

Centrifuge

LAB 2

"Specimen Processing" Lab

LAB 1

"No Template" Lab

workbench

Pass-through window

workbench

NA extraction equipment

sink

Ice machine

Eye wash

Lab coat rack

Lab coat rack

Lab coat rack

Lab coat rack

Eye wash

Ice machine

sink

workbench

workbench

LAB 4

"Post-amplification" Lab

LAB 3

"Nested PCR" Lab/Amplification Lab

Centrifuge

Sequencer

Gel electrophoresis area

sink

PCR machines

PCR machines

workbench

BSC: bio-safety cabinet


Three lab setup option 1

Three-Lab Setup (Option 1)

LAB 1

"Specimen Processing" Lab

Specimen receipt & storage

RNA extraction and 1st round PCR set-up

SPECIMENS

Frozen plasma

Whole blood

DBS

Master mix preparation

LAB 3

"Post-amplification" Lab

Gel electrophoresis

Sequencing reactions

Capillary electrophoresis

LAB 2

"Amplification" Lab

1st round amplification

2nd round amplification

2nd round PCR set-up

In dead-air cabinet with dedicated pipettes


Pcr workstations dead air cabinets

PCR Workstations (Dead-air Cabinets)


Three lab setup pre fabricated pcr laboratory

Three-Lab SetupPre-Fabricated PCR Laboratory

3

2

1

http://www.lifesci.com/xcart/product.php?productid=16&cat=1&page=1


Three lab setup option 2a

Three-Lab Setup (Option 2A)

LAB 2

"Specimen Processing and PCR" Lab

Specimen receipt & storage

RNA extraction and 1st round PCR set-up

SPECIMENS

Frozen plasma

Whole blood

DBS

1st round amplification

2nd round PCR set-up

LAB 1

"No Template" Lab

Reagent storage and

Master mix preparation

LAB 3

"Post-amplification" Lab

2nd round amplification

Gel electrophoresis

Sequencing reactions

Capillary electrophoresis

In dead-air cabinet with dedicated pipettes


Three lab setup option 2b

Three-Lab Setup (Option 2B)

SPECIMENS

Frozen plasma

Whole blood

DBS

LAB 2

"Specimen Processing" Lab

Specimen receipt & storage

RNA extraction and 1st round PCR set-up

LAB 1

"No Template" Lab

Reagent storage and

Master mix preparation

LAB 3

"Post-amplification" Lab

2nd round amplification

Gel electrophoresis

Sequencing reactions

Capillary electrophoresis

1st round amplification

2nd round PCR set-up

Option 2B is the preferred configuration if no nested PCR is performed

In dead-air cabinet with dedicated pipettes


Two lab setup

Two-Lab Setup

LAB 1

"Specimen Processing" Lab

Specimen receipt & storage

RNA extraction and 1st round PCR set-up

SPECIMENS

Frozen plasma

Whole blood

DBS

Master mix preparation

LAB 2

"Post-Amplification" Lab

1st round amplification

2nd round amplification

Gel electrophoresis

Sequencing reactions

Capillary electrophoresis

2nd round PCR set-up

In dead-air cabinet with dedicated pipettes


Example 2 lab layout

-80°C

freezer

-20°C

freezer

-20°C

freezer

-80°C

freezer

BSC

BSC

Example 2-Lab Layout

Ice machine

Centrifuge

LAB 1

"Specimen Processing" Lab

Dead air cabinet

NA extraction equipment

sink

Eye wash

Lab coat rack

Lab coat rack

Eye wash

Ice machine

PCR machines

Dead air cabinet

workbench

LAB 2

"Post-amplification" Lab

Centrifuge

Sequencer

Gel electrophoresis area

PCR machines

BSC: bio-safety cabinet


Discussion

Discussion

  • What is the ideal set up for a molecular lab?

  • What compromises can—and cannot—be made when the ideal is not possible?


Other workflow considerations

other workflow considerations

What else can we do to minimize the chances of contamination in the lab?


Personnel

Personnel

  • Perform pre-amplification work before entering post-amplification areas

  • Incorporate this (and all related contamination-control policies) into SOPs and/or lab policies


Lab coats

Lab Coats

  • Disposable or dedicated for each area

  • Color-coded if possible


Hiv drug resistance training

SOP

  • Make copies of SOPs available in each lab to prevent upstream traffic


Storage freezer locations

Storage Freezer Locations

  • Specimen logs, access to computerized inventory, etc. in pre-amplification area

  • RT-PCR reagents should be stored in separate freezers from specimens.


Application of principles

application of principles

What are examples of good and poor workflow?

What can we do, in our labs, to get the best possible lab set up and workflow?


Example 1 what works what doesn t

Example 1: What works? What doesn’t?


Example 2 what works what doesn t

Example 2: What works? What doesn’t?


Example 3 what works what doesn t

Example 3: What works? What doesn’t?


Example 4 what works what doesn t

Example 4: What works? What doesn’t?


Example 5 what works what doesn t

Example 5: What works? What doesn’t?


Example 6 what works what doesn t

Example 6: What works? What doesn’t?


Discussion1

Discussion

  • What are examples of good and poor workflow?

  • What can we do, in our labs, to get the best possible lab set up and workflow?


Small group activity

Small Group Activity

  • What can we do, in our labs, to get the best possible lab set up and workflow?


Summary

Summary

  • Principles of Lab Set Up

  • Other Workflow Considerations

  • Application of Principles


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