Lab Training Protocol Powerpoint Continue

Lab training protocol

1. Basic Requirements
Research involving recombinant DNA is closely
regulated by the National Institutes of Health (NIH).
Research involving biological agents is also regulated
by the NIH, the Centers for Disease Control and
Prevention and the State of California.UC Berkeley
requires a Biological Use Authorization (BUA)
approved by the Berkeley campus Committee on
Laboratory & Environmental Biosafety (CLEB). CLEB
works with campus Biosafety Officer Bob Hashimoto,
who can provide additional guidance
([email protected] or 643-6562).

2. Basic Requirements (Cont’d)
All work involving recombinant DNA/biological
agents - regardless of funding source - must adhere
to the mandatory NIH “Guidelines for Research
Involving recombinant DNA Molecules” found at
http://oba.od.nih.gov/rdna/nih_guidelines_oba.html

3. Purpose of This TrainingSection IV-B-7-d-(2) of
the NIH Guidelines
The NIH requires that the PI provide lab-specific
biosafety training on the hazards of the specific
experimental procedures used in their laboratory to
members of their research group. One way to
document this training is to record it in the minutes
of a group meeting, listing all participants.

4. Contents of This Training
CLEB Administrative Requirements
What are the NIH Guidelines?
Role of the Investigator/Lab Staff
Description of the Experiment-BUA
The Agents Used
The Template
Risk Assessment/Biosafety Level
Good Work Practices
Personal Protective Equipment
Engineering Controls
Waste Disposal/Decontamination
Emergency Procedures

5. CLEB Administrative Requirements
CLEB requires the following from every PI approved
to use recombinant DNA:
Train all research group members on lab-specific hazards and
procedures
Report incidents involving recombinant DNA immediately to
EH&S
Submit a BUA amendment for any change in personnel or
Scope of Work

6. What are the NIH Guidelines?Key Sections of the NIH
Guidelines
Appendix B-List of Agents
Section III-Types of Experiments involving
recombinant DNA (III-D-1 and III-E-3)
Appendix G-Level of Containment (BSL-2 and
ABSL-1)
Appendix K-Large Scale
Appendix M-Gene Transfer/Human Subjects
Appendix P-Transgenic Plants
Appendix Q-Transgenic Animals
Section IV-Responsibilities of PI, IBC

7. PI RESPONSIBILITIESSection IV-B-7: ROLE OF THE PI
On behalf of the institution, the Principal Investigator
is responsible for full compliance with the NIH Guidelines
for any recombinant DNA research.
The PI must:
Not initiate or modify recombinant DNA research which requires
CLEB approval until that research has been approved by CLEB
and has met all other requirements of the NIH Guidelines.
Report any significant problems, violations of the NIH
Guidelines, or any significant research-related accidents and
illnesses to the Biological Safety Officer and CLEB.

8. OTHER PI RESPONSIBILITIES
Responsibility NIH Guideline Reference
Train Lab Staff (Micro technique)Section IV-B-7-a, d
Ship Agents Safely Section IV-B-7-a
Follow Emergency Plans Section IV-B-7-a
Notify NIH of novel host/vectorsSection IV-B-7-b
Update Protocol Section IV-B-7-c
Notify IBC/CLEB of incident Section IV-B-7-e
Make Protocol AvailableSection IV-B-7-d
Offer Medical SurveillanceSection IV-B-7-d
Supervise Lab Members Section IV-B-7-e
Maintain Safety EquipmentSection IV-B-7-e

9. Description of the Experiment-BUA
Research Objective
Section I – Type of experiments
Section II – Personnel
Section III – Location of Research Experiments
Section IV – Physical Containment Equipment
Section V – Safety Evaluation
Section VI – Scope of Work Narrative
Attachement II- Section A: Recombinant DNA
Attachement II- Section B: Biohazardous Agents and Toxins
Worksheet I – Recombinant DNA Experiments Questionnaire
Worksheet 2 – Animal Experiment Questionnaire
Please see BUA for details

10. Description of the Experiment-BUAThe
Agents Used
Host cells: HEK-293
E. Coli
Fixed monkey tissue
Adeno Associated Virus
Adenovirus Type 5
MAG

11. Description of the Experiment-BUAThe
Risks That May Affect Containment Level
These are some of the common experimental risks
associated with our research:
Route(s) of exposure
aerosol generating procedures
Procedures involving sharps
the use of vectors
The use of animals
Our containment level:
BSL-2 for in-vitro work involving use of RG2 agents
ABSL-1 for housing and BSL-2 for in-vivo procedures

12. Description of the Experiment-BUAThe
Risks That May Affect Containment Level
These are the symptoms of exposure with the agents we use
(Adenovirus):
fever, rhinitis, pharyngitis, tonsillitis, cough and conjunctivitis
Most adults had a natural adenoviral infection early in
childhood, which results in immunity to subsequent adenoviral
infections.

13. Our Recombinant DNA Experiment Template
Agent Characteristics:
Routes of Exposure:
Host: E. coli, rAAV, HEK-293 cells
Vector: P-Helper (Adenoviral helper genes), AAV Helper (AAV rep and cap genes), rAAV
plasmid (promoter and gene of interest flanked by Inverted Terminal Repeats of AAV)
Nature of inserted sequences: promoter specific for cells and gene of interest, AmpR
Source of inserted sequences: mus musculus, homo sapiens, rattus norvegicus,
aequorea victoria, anthozoa, chlamydomonas reinhardtii
Types of manipulation:
Attempt to express foreign gene: yes, AmpR, GFP, mCherry, GCaMP5
Protein produced: GFP (tracking gene), mCherry (tracking gene), iGluR6 (ion channel),
GDNF (neurotrophic factor), CNTF (growth factor), XIAP (inhibitor of apoptosis), RO4
(G-protein coupled receptor), ChR (ion channel), FGF (Growth factor), RdcvF (survival
factor), RS1 (Structural extracellular protein), GcaMP5 (genetically encoded calcium
indicator)
Containment/Biosafety Level: BSL 2 for in vitro experiments, BSL 1 for in vivo
experiments
Section of Guidelines: Section III-D-1

14. Risk Assessment/Biosafety LevelGood
Work Practices
Wash Hands Frequently
Use Personal Protective Equipment and
Engineering Controls
Minimize Use of Sharps
Do Not Eat, Drink, or Smoke in the lab
Disinfect Work Areas

15. Risk Assessment/Biosafety LevelPersonal Protective Equipment
The following personal protective equipment must
be worn when handling agents in the tissue culture
room and the biosafety cabinet:
Latex or nitrile gloves
Lab coat. . In room 234 Barker Hall (cell culture)
disposable Tyvek labcoats are provided.
Safety glasses

16. Risk Assessment/Biosafety LevelEngineering Controls
The following engineering controls must be used
whenever handling agents in the tissue culture
room:
Biosafety cabinet
Sink for Hand Washing
Mechanical pipettes
Sealed vials
Sharps containers/Broken Glass box
Eyewash/deluge shower (use only in an emergency)

17. Waste Disposal/Decontamination
Liquid waste will be treated with 10% household
bleach and allowed to set for 20 minutes and
drain-disposed. Solid red bag wastes will be placed
in a red-bag lined bin and transferred to the
Koshland Building, Room LL32 medical waste
storage area for pick up and disposal.
Work areas must be disinfected with dilute bleach
and 70% ethanol.

18. Emergency Procedures

In the event of a laboratory accident:
Tend to injured person
Notify the PI or the lab supervisor; inform the PI
IMMEDIATELY if recombinant DNA was involved so
that CLEB and the Biosafety Officer can be notified
within 24 hrs.
Initiate clean up procedures.
Notify EHS, if the PI is not available, 642-3073 or
642-3333 (After Hours)
Seek medical attention at Tang or the Alta Bates ER at
2450 Ashby Avenue after hours.

19. Conclusion
This training is designed to inform you of the
NIH requirements regarding use of recombinant
DNA, and our lab’s implementation of the NIH
requirements. All recombinant DNA experiments
require BUA approval by CLEB and any changes
require an approved amendment to the BUA. If
you have any questions on this training, let me
know, and I will contact the Biosafety Officer for
assistance.

20. Required Trainings
Laboratory Safety for New Graduate Students and Employees
Biosafety in Laboratories – presence class
Biosafety in Laboratories Refresher – refresher every three years
Hazardous Materials Spill Response Training
  
Hazardous Materials Spill Response Refresher – yearly refresher
Bloodborne Pathogens Online Training (when working with viral production or cell
culture) - through Blu Portal every year!
Basic Safety for New NAF Useres (
http://www.olac.berkeley.edu/Unnamed%20Site/Classes%20&%20Training/Inde
x1.html
)
CITI on-line training (http://www.acuc.berkeley.edu/training.html) - the website gives
instructions how to do that (repeat every 5 years)
NAF Basic Safety Refresher (Northwest Animal Facility)- refresher every year
For Chemical/Hazradous waste go to
http://ehs.berkeley.edu/component/content/article/28-hm/279-new-hazardous-w
aste-program-hwp.html
(first time log in takes you to a training)
EHS102