Mission

InnovaBio positively impacts the community by giving students real-world research experience working on projects for life science companies in need of innovative technical solutions for research problems. InnovaBio offers scientific expertise and a low-risk financial environment that benefit both the partner enterprise and the students participating in the scientific work. Our mission is to mentor the next generation of Biotechnology Graduates and simultaneously assist local scientific and biotech enterprises in developing research projects.

To assist yourself and your team in fulfilling the mission, please do

the following steps:

Step1: Be Aware of your experimental plan: know what it is

and where to find protocols.

Step2:Understand the steps within the protocols or

intended experiments, and how theexperiments fit into the

overall project goals.

Step3:Be confident inwhat you are about to embark on is the

correct experiment for accomplishing the goals of the project.

Step4:Act. Conduct the experiment and analyze results

to know what you should do next

Training Packet

2016

Name: ______

Objectives:

-Become familiar with equipment and reagents in the lab

-Review how to make solutions

-Become familiar with PubMed

-Become familiar with planning, conducting, and recording experiments.

Please read through entirely:

-Purify, quantify, and digest plasmid from bacterial cultures.

-Induce and document production of recombinant protein in E. coli.

-Learn the importance of experimental controls.

-Become familiar with Good Laboratory Practices.

Training

-Lab Notebooks

-Pipetting

-Autoclave

-PubMed

-Making solutions

Evaluations and “Grading”:

-You will be evaluated on your performance in the lab as an employee of InnovaBio. We ask you to report an honest assessment of yourself using the guidelines found in the syllabus. You will be sent a link through Google Groups. Please complete these in a timely fashion. The grading criteria can be found on page 14. Please review it.

-You will need to fill out an evaluation upon completion of the training packet if not sooner.

-All significant dates and deadlines for evaluations, presentations, and assignments will be noted on the Google Calendar.

PubMed and PubMed Central

PubMed is an online site set up and run by the government and the U.S. National Library of Medicine. It contains sources from MEDLINE, science journals, and biomedical articles that date back to the 1950’s. The site contains information on genomes, proteins, nucleotides, and much more. The site is useful in that it provides researchers with up to date information about genes, proteins, and other research projects going on. Some of the journals and articles within PubMed are free, but many are abstracts of certain articles that can be purchased to view. PubMed can be accessed by going to

PubMed Central is an archive of life science journals, biomedical articles, and other informational literatures that can be found on PubMed, but are full text and free to the public. PubMed Central can be accessed through the PubMed website or by going to

Your assignment is to research a topic of your choice on any of these two websites and print out a primary research paper that seems appealing to you. Read through the article and write a 1 page paper on the paper you have read. First, summarize the paper using the scientific format (without using personal pronouns). Then, include what you learned, did not know, and want to learn more about. This paper is due on the day you complete the training packet.

What you did / Name on tube cap / What indicates / Name on side
of the tube
Labeling DNA samples /
You purified from 5 different E. coli colonies a pET21a(+) plasmid with hmgb1 cloned in it (hmgb1 is the gene of interest, also called insert). You did the plasmid prep on April 4th, 2010. / pHMGB1
1
4/4/10 / Indicates hmgb1 cloned into plasmid “p” was prepped on April 4th, 2010 (you should specify on your book the identity of plasmid “p”).
On this date, your book should have an entry mentioning you prepped plasmid pHMGB1 from several different colonies. / Your initials.
The concentration of the plasmid.
On April 4th, 2010, you amplified then purified a PCR product. The gene you are working with is hmgb1 / HMGB1
pure PCR
4/4/10 / Indicates hmgb1 from a PCR experiment I conducted on April 4th, 2010, is in solution and that it is pure. On this date, your book should have an entry describing how you amplified and purified hmgb1. / Your initials.
If you can spare product, then determine and write its concentration.
On April 4th, 2010, you digested your hmgb1 PCR product with enzymes HindIII and XbaI. / HMGB1
PCR
HindIII/XbaI
4/4/10 / Indicates PCR product hmgb1 was digested with enzymes HindIII and XbaI. Your book should have an entry describing how you digested your PCR product on the indicated date. / Your initials.
What you did / Name on tube cap / What indicates / Name on side
of the tube
On April 4th, 2010, you purified your hmgb1 PCR product digest. / HMGB1
dig HindIII/XbaI
pure
4/4/10 / Indicates you purified the PCR product you digested with HindIII and XbaI. On the indicated date, your book should have an entry mentioning you purified your hmgb1 PCR product digest. / Your initials.
You digested pHMGB1-1 with enzymes HindIII and XbaI on April 4th, 2010. / pHMGB1-1
HindIII/XbaI
4/4/10 / Indicates plasmid pHMGB1, which comes from colony 1, was digested with restriction enzymes HindIII and XbaI on April 4th, 2010. On this date, your book should have an entry describing how you prepared the digestion(s). / Your initials.
On April 4th, 2010, you ligated digested plasmid and a digested DNA fragment. / Name of plasmid
Name of DNA fragment
lig
4/4/10
Could go like this:
pET21a(+)
hmgb1
lig
4/4/10 / Indicates you ligated plasmid pET21a(+) with hmgb1 on the mentioned date. On that date, your book should have an entry describing how you set up the ligation reaction. / Your initials.
Labeling protein fractions
On Sep. 15th, 2010, you prepared cells in SDS loading dye to run a SDS-PAGE for analysis of gene expression / Name of protein product
9/15/10
exp 1 / “exp 1” indicates that the gene expression was carried out under condition “1”, which should be explained in detail in your lab notebook. There can be as many “numbers” as testing conditions for gene expression. You may have several different tubes, each with the protein product manufactured at different expression conditions. / Your initials. The absorbance value of the cell culture before it was spun down.
On Aug. 24th, 2010, you lysed cells and separated the soluble from the insoluble fraction by transferring the soluble fraction to a new tube. / Name of protein product
8/24/10
Sol
con 1 / “Sol” and “Insol” mean soluble and insoluble, respectively. “con 1” indicates the lysis conditions employed to obtain the samples. Detailed information describing the lysis conditions should be written in your lab notebook. / Your initials. Information to trace this back to expression conditions.
Name of protein product
8/24/10
Insol
con 1
You treated sample “insol, con 1” with urea and/or other detergents, and separated the urea soluble fraction from the urea insoluble fraction by transferring the former to a new tube. You did it on Aug. 25th, 2010. / Name of protein product
8/25/10
U-Sol
U-con 1 / “U-sol” and “U-insol” mean urea-treated soluble and insoluble fraction, respectively. “U-con 1” indicates the conditions employed under the urea treatment. Detailed information describing the protein solubilization treatment with urea should be written in your lab notebook. / Your initials. Information to trace this back to expression conditions.
Name of protein product
8/25/10
U-Insol
U-con 1
You initiated the purification of your protein by loading it on a Ni2+ IMAC (HisTrap) or any other of our columns (HiTrap Q, Sephacryl S-200 HR, etc). You collected fractions (1…etc.) from your column as proteins were being eluted. This was done on Aug. 25th, 2010. / Name of protein product
Ni
8/25/10
1 / “Ni” indicates that the sample was eluted from a Ni2+ IMAC (HisTrap) and “1” is the fraction number. This number should match the AKTA FPLC elution pattern for the run. This labeling system can be used regardless of the column from which the protein is being eluted. / Your initials.
You continued the purification of your protein by loading it on a Q HiTrap. You collected fractions (1…etc.) from your column as proteins were being eluted. This was done on Aug. 26th, 2010. / Name of protein product
Q
8/25/10
1 / ”Q” indicates that the sample was eluted from a Q HiTrap and “1” is the fraction number. This number should match the AKTA FPLC elution pattern for the run. This labeling system can be used regardless of the column from which the protein is being eluted. / Your initials.

Exercises

Fill in the blocks

What you did / Name on tube cap / What indicates / Name on side of the tube
pET21-a
VG1-1
Nde1/HindIII
On Sep. 12th, 2010, you set up a PCR experiment and obtained a PCR product using primers 1 and 2 and condition 3. The name of the PCR product is A LOT.

Lab Notebooks

You will write all procedures into your lab notebook provided by InnovaBio. You must write out the experimentbefore you start handling any reagents. Every experiment should be recorded whether “it worked” or “didn’t work”. All lab notebook entries need to follow the following guidelines:

  • Name - List names of all who participated.
  • Title - Title the lab procedure you are doing.
  • Date - Write the date or dates of the experiment
  • Project and book numbers- Indicated in provided space. (Training Packet=Project No.1)
  • Purpose - Write out why you are doing the experiment
  • Materials - List all the materials you usewith lot numbers for reagents that have them.
  • Protocol - List all the procedures you will go through to complete the experiment. Give specific details and show any calculations.
  • Observations - Write out everything that you observed throughout the experiment. List any modifications made in the methods you listed, all unexpected results, and anything that would be useful to someone following the same experiment. Also place any graphs, tables, or printouts.
  • Any corrections must be indicated by crossing out the incorrect part with a single line, writing the correct information with your initials and the date.
  • Conclusion - From your observations, conclude whether the experiment worked or not. Explain why the experiment did or did not work and back your conclusion up referencing your methods, observations, or other materials.
  • References - Make sure to write in any references used in your protocol. Did you use any papers, SOP’s, or other materials? Reference all sources that you used so that someone else can find the information.
  • Each day you and a witness will sign and date the pages of the lab book.

Good Laboratory Practices

Good laboratory practices (GLP) were developed as a set of quality standards to ensure the validity of and confidence in experimental results derived from non-clinical research. The major components of GLP centeron Quality Assurance (QA) and include Standard Operating Procedures (SOPs), statistical procedures for data evaluation, instrument validation, reagent and materials certification, specimen and sample tracking, and documentation and maintenance of records. While this is only a partial list, it is evident that applying these standards to most lab work and research could prove quite beneficial. Use of SOPs will ensure that a variety of procedures are done consistently and reliably. Ensuring that the equipment you’re using is good working order -and properly calibrated if necessary- and leaving that equipment clean and in good working order for the next user will benefit the lab as a whole and bolster confidence in results generated with that equipment. Careful monitoring and documentation of stock solutions will promote confidence in their use and allow problems to be identified readily should they arise. When setting up experiments, be sure all necessary controls exist such that your experiments can be appropriately and confidently interpreted. Careful note taking and documentation of experimental procedures and results (your lab notebooks) will ensure confidence in and reproducibility of those results. In the lab, certain stock solutions will have log sheets associated with them such that appropriate SOPs are utilized and the date they were made and the person who made them are apparent. You will all be making these solutions at some point, so adhering to these simple guidelines should allow you to have confidence in solutions made by yourself as well as others and additionally ensure consistency in their use. Some equipment in the lab may have logs as well but will at least have guidelines for use which will include shut down procedures such that the equipment is clean and good working order for the next user. Importantly, take time to read over the lab notebooks section of the training packet; the guidelines outlined will ensure that your experiments are appropriately documented. GLP standards exist in many of the work environments you’ll potentially enter and becoming familiar with them now will only facilitate the ease with which you assimilate them later on. Additionally, some of the more basic standards will definitely improve the quality of your work and allow you to have confidence in the outcomes. Take a minute to look up GLP standards in order to familiarize yourselves with some of the terminology and practices.

Risk Assessments

In addition to knowing and understanding the laboratory rules and expectations as outlined in the safety orientation, it is extremely important to be aware of any hazards associated with the specific experiments you are doing. Hazards can be identified by completing a risk assessment form, like the example provided below.

Assignment

Choose an experiment from the training packet and complete a risk assessment form. Forms can be found in the lab or on the lab computer. Chemical safety information can be found in the material safety data sheets (MSDS) in the lab or online. Please consider the following questions when completing the risk assessment:

Description of task/experiment

-What experiment are you doing, and does it require special conditions (heat, gas etc.)?

Hazard identification: equipment

-What equipment will you be using? Are there hazards associated with it?

Hazard identification: chemical

-What reagents are you using, and what are their chemical hazards?

-What is the National Fire Protection (NFPA) fire diamond? What do the colors/numbers mean?

-What role does each reagent play in the experiment?

Safety controls; precautions; waste disposal

-What protective equipment will you need?

-How will you dispose of any waste?

Name / Date
Location / Phone
Description of task/experiment
Repetitive task Services used: Water Power Gas Other Temp100oC Pressure…
Preparation and use of agarose gel for DNA purification.
Melt agarose in TAE buffer in a conical flask by microwaving. Once the gel is cool, add ethidium bromide and pour into gel casts. Once the gel has set, load DNA samples, run the gel and visualize the DNA using the gel dock.
Hazard Identification: Equipment
Microwave – burn hazard
Electrophoresis Power Pak power supply – high voltage.
Gel Doc – UV light
Hazard Identification: Chemical
Name / Health / Fire / Reactivity / Other / Use/purpose of reagent/notes
50X TAE
(Tris-acetate-EDTA) / 0 / 0 / 0 / Gel preparation and running buffer. Tris-acetate provides ions to conduct the electrical current to aid DNA mobility, and maintains the pH. EDTA chelates divalent metal ions to inhibit the action of DNase on the DNA samples.
agarose / 0 / 0 / 0 / Agarose is a polymeric polysaccharide that forms pores upon setting in the gel form. Agarose density determines the pore size. These pores act like a sieve and allow the DNA to be separated by size.
Ethidium bromide / 3 / 0 / 0 / Ethidium bromide is a fluorescent dye used to visualize DNA in agarose gels by intercalating between the base pairs of the two DNA strands.
Safety Controls; Precautions; Waste Disposal
Use personal protective equipment (PPE): lab coat, gloves, and safety glasses/goggles.
Use caution when microwaving glassware as the glass and contents will be hot. Use heat-resistant gloves to carry glassware.
Be aware of general electrical shock hazards when using the PowerPak power supply.
When using UV light source, protect all skin from UV exposure by using gloves, lab coat etc. Protect eyes by using appropriate face shield.
Ethidium bromide is a mutagen: use PPE when handling. Use designated chemical waste; do not dispose down the drain.

Lab Benches

You will be provided a lab bench. You are responsible for keeping your bench clean and for ensuring that all equipment is in proper working order. You will test your pipettes for proper function using the Artell. Remove, from your bench, any unlabeled bottles or old reagents. Clean your bench and overhead shelves of any dust or residue. Acquire new autoclaved water and LB broth from the cabinet. Label them with your initials and the date.

Every day when you arrive and before you leave, sanitize your bench by clearing it of any equipment, emptying biohazard buckets, and wiping the bench top with 70% ethanol. This will help to prevent contamination coming from those who may have used your bench while you were away.