HIV Drug Resistance Training
Module 4:
Molecular Lab Set-up
and Workflow
1
Topics



Principles of Lab Set Up
Other Workflow Considerations
Application of Principles
2
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.

3
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?
4
Amplification of PR/RT Sequences
Reagent
Preparation
Specimen
Preparation
Amplification
Sequencing
0
105
1010
1012
Copy #
5
Genotyping Assay Procedures
Patient virus
RNA Extraction
cDNA synthesis (RT)
Positive and
negative controls
cDNA amplification (PCR)
Sequencing reactions
Positive control
Electrophoresis
Data Analysis (base calling)
Report (sequence, mutations, interpretation)
6
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
LAB 1
"No Template" Lab
2nd round amplification
Gel electrophoresis
Sequencing reactions
Capillary electrophoresis
Reagent storage and
Master mix preparation
LAB 3
“1st Round PCR” Lab
1st round amplification
2nd round PCR set-up
7
-20°C
freezer
BSC
BSC
LAB 2
Ice
machine
workbench
"No Template"
Lab
Lab coat rack
NA extraction
equipment
Centrifuge
sink
Lab coat rack
Eye
wash
sink
Eye
wash
workbench
Ice
machine
LAB 4
-20°C
freezer
PCR
machines
workbench
Lab coat rack
"Nested PCR"
Lab/Amplification
Lab
workbench
"Specimen
Processing" Lab
-80°C
freezer
Passthrough
window
LAB 3
sink
Ice
machine
LAB 1
Lab coat rack
-20°C
freezer
-20°C
freezer
-80°C
freezer
workbench
sink
Centrifuge
Example of a Four-Lab Layout
(Nested PCR/In-House Assays Only)
"Postamplification" Lab
Sequencer
Gel electrophoresis area
PCR
machines
8
BSC: bio-safety cabinet
Three-Lab Setup (Option 1)
SPECIMENS
Frozen plasma
Whole blood
DBS
LAB 1
"Specimen Processing" Lab
Specimen receipt & storage
Master mix preparation
RNA extraction and 1st round
PCR set-up
LAB 3
"Post-amplification" Lab
LAB 2
"Amplification" Lab
Gel electrophoresis
Sequencing reactions
Capillary electrophoresis
1st round amplification
2nd round PCR set-up
2nd round amplification
In dead-air cabinet with dedicated pipettes
9
PCR Workstations (Dead-air Cabinets)
10
Three-Lab Setup
Pre-Fabricated PCR Laboratory
3
2
1
11
http://www.lifesci.com/xcart/product.php?productid=16&cat=1&page=1
Three-Lab Setup (Option 2A)
SPECIMENS
Frozen plasma
Whole blood
DBS
LAB 2
"Specimen Processing and
PCR" Lab
Specimen receipt & storage
RNA extraction and 1st round
PCR set-up
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
12
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
Option 2B is the preferred
configuration if no nested PCR
is performed
LAB 3
"Post-amplification" Lab
1st round amplification
2nd round PCR set-up
2nd round amplification
Gel electrophoresis
Sequencing reactions
Capillary electrophoresis
In dead-air cabinet with dedicated pipettes
13
Two-Lab Setup
SPECIMENS
Frozen plasma
Whole blood
DBS
LAB 1
"Specimen Processing" Lab
Specimen receipt & storage
Master mix preparation
RNA extraction and 1st round
PCR set-up
LAB 2
"Post-Amplification" Lab
1st round amplification
2nd round PCR set-up
2nd round amplification
Gel electrophoresis
Sequencing reactions
Capillary electrophoresis
14
In dead-air cabinet with dedicated pipettes
Ice
machine
-20°C
freezer
BSC
BSC
-80°C
freezer
Centrifuge
Example 2-Lab Layout
LAB 1
Dead air
cabinet
NA extraction
equipment
Lab coat rack
Lab coat rack
PCR
machines
Eye
wash
Dead air
cabinet
Ice
machine
LAB 2
-20°C
freezer
Centrifuge
workbench
Eye
wash
sink
-80°C
freezer
"Specimen
Processing" Lab
"Postamplification" Lab
Sequencer
Gel electrophoresis area
PCR
machines
15
BSC: bio-safety cabinet
Discussion


What is the ideal set up for a molecular lab?
What compromises can—and cannot—be made
when the ideal is not possible?
16
other workflow considerations
What else can we do to minimize the chances of contamination in
the lab?
17
Personnel
Perform pre-amplification work before entering
post-amplification areas
 Incorporate this (and all related contaminationcontrol policies) into SOPs and/or lab policies

18
Lab Coats


Disposable or dedicated for each area
Color-coded if possible
19
SOP

Make copies of SOPs available in each lab to
prevent upstream traffic
20
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.

21
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?
22
Example 1: What works? What doesn’t?
23
Example 2: What works? What doesn’t?
24
Example 3: What works? What doesn’t?
25
Example 4: What works? What doesn’t?
26
Example 5: What works? What doesn’t?
27
Example 6: What works? What doesn’t?
28
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?
29
Small Group Activity

What can we do, in our labs, to get the best
possible lab set up and workflow?
30
Summary



Principles of Lab Set Up
Other Workflow Considerations
Application of Principles
31