Monday, June 30, 2008
Week 1: SIP sharing
Hi everyone! I will be the first in my group to share about what I have learnt during my first week of attachment. Attachment HERE is very fruitful because I get to learn from my mentor, we have a comfortable environment for work and the laboratory is very well equipped. =)
I am attached to a research laboratory which deals with delivery of proteins, genes and drugs into cells. The particular laboratory I am working in focuses on delivery of genes into mammalian cells, hence we are required to carry out a lot of Molecular Biology and Mammalian Cell techniques. My MP would also be on the delivery of certain genes into mammalian cells to get a certain desired outcome.
The delivery of genes is not as straight forward as I thought, because we have to keep constructing new viruses for each new type of gene we want to deliver! The virus vector is constructed to carry the desired gene so that it can be transferred to the mammalian cells via transduction.
For the whole of the first week, I have been learning how to construct viruses and how to measure the titer of the viruses that my mentor has already constructed. I am still learning how to construct the virus as it involves many steps. So far, I have only performed up to the digestion of the vector and the insert (step 6).
Outline on how to construct a Virus (The main points are in red, the details are for those who may be more interested in how the step goes about).
1) Culture the cells containing the desired gene that we want to package into the virus, on a tissue culture flask. Cells will adhere onto the treated surface of the flask, spread and grow.
2) Maintain the cells in healthy condition and prepare a suspension culture (by trypsinization) for counting and DNA extraction. Trypsin detaches the cells from the surface of the flask by breaking down protein that binds cells to the flask surface. Counting of cells is required as part of the DNA extraction protocol for maximal DNA extraction yield.
3) Use DNA extraction kit such as DNeasy® Tissue Kit to extract DNA from cell suspension and measure the concentration of DNA extracted.
4) Run the extracted DNA on agarose gel to confirm that the DNA is pure and check what is the kilo base pair of the DNA by comparing it with the DNA ladder marker.
5) Excise the band of DNA and dissolve gel to extract DNA using a gel extraction kit.
6) Obtain a suitable vector backbone to be ligated with the insert (gene) and perform
restriction digestion and ligation.
7) Transform cells with recombinant vector and select out the cells via antibiotic resistance and blue white colonies screening strategy.
8) Extraction of recombinant DNA using mini-prep kit.
9) Send the constructed recombinant DNA for sequencing to confirm the gene of interest is in the correct orientation for expression.
10) Tranfection of cells (to be infected by virus) with recombinant DNA. Transfection is a process of transferring DNA into the cells that are to be infected by the virus. The DNA is complex with some salts (calcium phosphate) from the transfection reagent that facilitate transport into the cells.
11) Virus is used to infect the transfected cells, and viruses will package these genes into their capsid as they replicate.
12) The virus titer is measured using plaque assay. (pfu/ml).
13) If the titer of the virus constructed is too low, we would need to amplify the virus.(another protocol).
14) Store these constructed virus stock in 4 degree Celsius cold room.
On the first day of my attachment, my mentor is already testing the efficiency of 2 types of viruses he has constructed months ago. He tested the efficiency of the viruses by measuring the virus titer (pfu/ml) using plaque assay. (Refer to below). Measuring the titer of the virus can be used for 2 purposes:
Determine the titer of virus constructed.
To determine the titer of virus stock that has been stored for a long time.
He needs to do this because the viruses are stored in 40ml centrifuge tube in the cold room at 4 degree Celsius, and overtime the virus titer will drop from 1 x 10^7pfu/ml (initial) to as low as 2.5 x10^6pfu/ml after 3 to 4 months. Measuring the titer is very critical because if the virus titer is too low, it will not be effective in infecting the cells and gene delivering efficiency consequently will be low.
Outline on how to perform Plaque Assay in a 6 well plate format
1) Harvest the cells that the virus can infect on the 6 well plates and incubate til cells are 50% confluence.
2) Perform serial dilution of the virus such as 10^-2, 10^-3, 10^-4, 10^-5, 10^-6, 10^-7 and 10^-8. in eppendorf tubes.
3) Remove medium from the cultured cells and add the diluted virus to the 6 wells and incubate to allow viruses to infect cells.
4) Remove the fluid (containing virus) in each of the well, as the viruses has already attached to the cells.
5) Add plaquing medium, which is a type of agarose gel containing medium to each of the well after the fluid is removed, and allow agarose gel to harden before incubating the plate of cells.
6) The function of the agarose gel is to immobilize the viruses so that it forms a clearing as it infects the cells.
7) These clearings (plaques) can be counted as plaque forming unit/ml, which is the titer of the virus.
That’s all for now! Thanks for reading my long entry! =)
Jean Leong
TG02
Monday, June 16, 2008
Subject: Laboratory Management & Quality Assurance
Topic: Safety Aspects in Clinical Laboratories & ISO 14K
Area: Bloodborne Pathogen (BBP) Standard
Aim: Minimise exposure to BBPs found in body fluids
Requirements to ensure compliance - Effective Training Program 1
- Exposure Control Plan 1,2
Form task categories to identify exposure potentials
HBV Vaccination
Develop procedures to handle exposure circumstances
Plan is reviewed & updated at least annually - Post-exposure Evaluation with Follow-Up 1
Documentation
Identification, testing, evaluation of source after consent taken - Engineering & work practice controls 3
Engineering: Biohazard hoods
Work practice:
PPE: gloves, laboratory coats, face protection 1,2
Handwashing procedures - SOPs. E.g. specimens secured in containers, disinfecting & decontamination, double gloves when handling body fluids 1
- Respiratory Protection 1
- Labelling
- Recordkeeping 1
(Number of words: 100)
References:
Book
1.Montgomery, Lynn.(1995).Health and safety guidelines for the laboratory. USA: Chicago, III: ASCP Press.
Websites
2.Occupational Safety & Health Administration. (2008). Bloodborne pathogens. - 1910.1030. Retrieved June 15, 2008, from
http://www.osha.gov/pls/oshaweb/owadisp.show_document?p_table=STANDARDS&p_id=10051
3.Lab Safety Supply Inc. (2008). Complying with OSHA's Bloodborne Pathogens Regulation, 29 CF 1910.1030. Retrieved June 15, 2008, from http://www.labsafety.com/refinfo/ezfacts/ezf105.htm
Lim Xin Ni TG02
Area: Hazardous Chemical Standard
This is a requirement by OSHA; standard aims to minimize exposure of personnel to hazardous chemicals. 1
Goal: educate workers on proper handling, storage and disposal. 1
-Scope and application
All employees who use Hazardous Chemical. 1
-Chemical hygiene plan
Protects employees from health hazards of chemicals and keeps exposure below specified limit. 1
-Employee information and training
As much literatures and trainings on methods of protection to chemicals. 1
-Medical consultation
Medical attention to signs and symptoms of potential exposure. 1
-Hazard identification; MSDS
MSDS: identify properties of chemicals produced by laboratory. 2
-Recordkeeping
Personnel medical consultations and accidents records. 1
(100 words)
Bibliography
Website
1. Environment, Health Ernest Drlandd Lawrence Berkeley National Laboratory. (2008) Chemical Hygiene and Safety plan: Chemical hazard: defination. Retrieved June 15, 2008 from http://www.lbl.gov/ehs/chsp/html/hazard_eval_.shtml
Book
2. Montgomery, Lynn.(1995).Health and safety guidelines for the laboratory. USA: Chicago, III: ASCP Press.
Jean Leong
TG02
Fire saftey concerns and disaster plans
-Fire codes
Explains the minimum requirements for fire prevention and safety. Adopted at facilities to ensure maximum prevention of fire.
-Signs
Let the people know where the escape routes and fire exits are(1)
-Storing flammables
Away from potential sources, by distance or by fire-resistant enclosures(2)
-Fire extinguishers and saftey equipment
Appropriate fire extinguishers should be placed at prominent places (E.g. exits) to act during fire. Inspection is important, check for any deficiencies and should be changed.(2)
-Fire drills and safety lectures
Should be practiced to train the laboratory staff and workers on various fire drill procedures. Preventative measures should be lectured(2)
-Disaster plans
Know the outline of the building and emergency exits(2)
(100 words)-info only
References
1.Wikipedia(2008), Fire codes, Retrieved on 16 June 2008
http://en.wikipedia.org/wiki/Fire_code
2.World Health Organization (1997), Safety in Health care laboratories, pg 33-38, Retrieved on 16 June 2008
Done by
M.Neela
Tg02
Area: ISO 14000 - ISO 14001 and ISO 14004
ISO 14000 environmental management standard1 helps minimize environmentally harmful waste produced by an organization, namely 14001 and 14004.
ISO 14001 is a management tools1 that helps find out and manage the environmental impact1 of its activities, products or services1, to constantly improve its environmental performance1, and to systematically achieve environmental objectives1 and targets set providing evidence of it to protect the environment.2
ISO 14004 provides detailed guidelines on development and implementation3 of the of environmental management systems (EMS)1. It describes the principles, systems and supporting techniques3 on EMS to fulfill the requirements and operate effectively according to standards.1
( total:100words)
Reference
1. International Organization for standardization. (2008). ISO 14000 essentials. Retrieved June 15, 2008 from http://www.iso.org/iso/iso_catalogue/management_standards/iso_9000_iso_14000/iso_14000_essentials.htm
2. Praxiom Reseach Group Limited. (2008). ISO 14001 2004 ENVIRONMENTAL MANAGEMENT STANDARD TRANSLATED INTO PLAIN ENGLISH. Retrieved June 15, 2008 from http://www.praxiom.com/iso-14001-2004.htm
3. David Sheppard. (2003) What is the ISO 14000 Series?. Retrieved June 15, 2008 from http://www.bsu.edu/web/dcsheppard/ISO/iso14000series.htm#_top
Teo Zhenling
TG02
Area: ISO 14000 Series
How does it work?
- Provides a general framework of multiple standards to tackle the organization's environmental policy, plans and actions.[1]
- Establishing a common reference for communication about environmental management issues between organizations and the public.[1]
What it achieves?
Internal objectives:
- Providing assurance to management that controls the organization’s processes and activities.[1]
- Providing assurance to the employees
Minimize negative effects of operation.
External objectives:
- Providing assurance on environmental issues to the community by complying with environmental regulations that supports the organization's claims about its policies, plans and actions.[1]
- Demonstrating conformity via several assessments
(Number of words: 99)
Reference:
1.International Organization for standardization. (2008). ISO 14000 essentials. Retrieved June 15, 2008 from http://www.iso.org/iso/iso_catalogue/management_standards/iso_9000_iso_14000/iso_14000_essentials.htm~
Debbie Tan
TG02
