Debate and discussion of any biological questions not pertaining to a particular topic.
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I've heard several time about people being "good at the bench" or "having good hands". Being a relatively new graduate student, and encountering problems with my experimentation skills, I ask all the guru's of mol biology to clarify those terms. What does a 'good laboratory worker' do that an 'ordinary' worker might have missed? How does he execute his experiments that he's always successful? Is it because of good planning? good execution? or experience? How should I prepare myself to become the super grad student. Please guide me.
Be ready to work hard, ask questions, even if they sound stupid, they might not, ie it is better to ask 1000 stupid questions than to destroy a 10K$ piece of equipment because you thought you knew how to use it (First hand exerience here.... ). Then read plan, think about control and troubleshooting even before you actually need them, and with abit of luck you might be seen as someon that is good at the bench.
But some people are naturally clumsy and will always be a catastrophe in the lab (there is such a master student in our lab, but last month he broke plenty of glassware, a mercury thermometer and a 3000$ piece of equipment...)
Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)
Thanks Canalon. I also read this book "At the bench" by Kathy Barker and wrote down things which were important for me. But nevertheless I thought it might be useful to somebody else also. I tried to attach the document, but I dont know why the forum did not allow me to upload ANY format (doc, rtf, txt, pdf). So I'm just pasting the whole thing below.
• Do your lab duties cheerfully and sincerely.
• ASK!!! Whenever you have a question. Never hesitate.
• Write down everything when somebody is giving instructions.
• Confess mistakes!!
• Clean up immediately after you have used something!
• Get demonstrations of every equipment in the lab, even when you know to use it. Things may be handled differently from lab to lab.
• Wash, return, clean up and turn off.
• For every equipment, know (1) how to operate it (2) ask if its turned off after use (3) if it needs to warm up before use (5) Is there a sign-up sheet?
• Daily: wipe down with 70% ethanol.
HOW TO SET UP AN EXPERIMENT
Set yourself as a careful and thorough investigator. Think about every experiment as a potentially publishable one. This way, you are careful about how you plan and execute the experiment.
1. Define the question: it should answer 1 or 2 questions, not try to solve every question of your project. Discuss with your colleagues about the question.
2. Design the experiment:
a. Background research: details and theories of the experiment, what techniques can be used, what results will be expected, what reagents are likely to work, and at what concentration they have been used. Start with a few classic papers in your field and learn about the details, then do a regular literature search, and also write/call to a particular PI in case more details are needed.
b. Experimental variables: what will be measured? What time points? Concentration?
c. Controls: every variable needs a control, and they should be repeated with every experiment.
d. Number of samples: duplicates for every experiment.
f. One may mistake the busyness & sense of importance one feels when doing an experiment as planning. THERE IS NO SUBSTITUTE FOR CAREFUL PLANNING!!!
3. Set up the experiment:
a. Get/prepare a protocol: best is to get from another investigator, or a book of protocols. The last desired option is to get from the materials and methods section of the published paper, as it may have missed several details.
b. Prepare reagents
c. Mental dry run
d. Physical dry run omitting crucial reagents, in case of tricky experiments
e. Observe someone else doing the experiment
4. Do the experiment: Follow the protocol every time, even if it’s done 50 times, with or without success in such a methodology.
5. Collect and analyze the data; discuss with someone.
6. Repeat it.
7. Common mistakes about setting up experiments:
a. Not thinking about the necessity of the experiment
b. Planning a huge and sloppy experiment
c. Forgetting to evaluate earlier experiments
d. Not thinking about every control to make the experiment interpretable
e. Not checking if every reagent is available before starting to do it.
Types of controls
1. Experimental controls: indicate whether the basic experimental procedures are working correctly.
2. Treatment controls: if the experimental treatment had an effect. If multiple treatments are there, the effects of individual factors must be controlled independently (if treatments with X and Y, a control would be treatment X and treatment Y separately)
a. Positive control
b. Negative control
3. Time points: a control for every variation in the time of the experiment. For example, if you are looking at the effect of factor X on cells 0, 15, 30 & 45 minutes, then every time point should have a control. Set up your time points such that harvesting of one sample is completed before harvesting the next. In case of sample jam, find a suitable “resting” place, like putting them on an ice bucket or something like that.
4. Zero time control: You must collect a sample (& control) as immediately after treatment as possible. Many times, it’s sensible to do this AFTER the normal time points, instead of doing side by side. For example if looking at the effect of factor at 0, 15, 30 & 45 minutes, first finish this and then do the zero time point control.
1. Read the protocol to see if it makes some sense to you, and imagine you doing it mentally. If you have ANY question, do not hesitate to email or call the author of the protocol/paper.
2. Change the protocol as you require and rewrite it
3. Prepare all reagents of the protocol and make sure that you have everything you need. EVERYTHING needs to be planned!!! (In case of kit, make sure you understand and write down how the kit works, for later troubleshooting)
4. Follow the protocol exactly the first time you do the experiment. You can modify the protocol later, with experience
5. Computerize your protocol, to make it personalized.
Interpreting the results
1. Did the experiment work? Check your controls: first check the experimental controls, and then check the positive controls (to check if the experiment was done properly), and then the negative controls (if its fine, you did it properly). If the negative control was positive, either the experiment was not planned properly or another variable asserted itself during the experiment
2. What are the results? Compared with the controls, minus the background, did you get an effect? How much effect?
3. What does the experiment mean?
4. Do other investigators understand your experiment?
5. Is the result repeatable? A key aspect of the experiment.
When your experiments don’t work
1. Is it a procedural problem? Check your equipment, check your protocol
2. Redo the experiment
3. If the problem recurs, redo only that part of the experiment that is in question
4. When you have identified a probable source of the problem, do a small experiment to see whether the problem is fixed. Don’t be tempted to jump in and repeat the original experiment yet- wait until the result has been explained
5. When you cannot find the source of trouble, ask advice and repeat it.
MAINTAINING A LABORATORY NOTEBOOK
Ask PI about how they expect the lab notebook to be maintained
The record of every experiment should contain
a. The date
c. Protocols, or references to the protocols used
d. EVERYTHING THAT HAPPENS, AND THAT DOESN’T HAPPEN, IS DATA!! Record everything.
e. At the end of accumulation of all data, write a one-sentence summation of the results of the experiment. Include aberrations, and any comments as to why the experiment may or may not have worked
f. Common things that are missed: refer book
g. Solicit feedback on your data and your plans.
When you meet your PI
1. Regularly meet him/her. Important.
2. Give a brief introduction
3. Label every figure and every sample clearly, those which are important and those which are not
4. Say your conclusion, and give the next steps
5. Follow-up on the meeting.
Which water to use
1. Laboratory grade water: water treated by reverse-osmosis or distillation. Adequate for making buffers
2. Reagent grade water: the lab grade water is further purified through distillation or deionization. Can be used for cell culture and biochemical reagents. This is plentiful for making buffers
3. Ultrapure reagent grade water: the reagent grade water further purified for specific purposes.
4. Many institutions have “house distilled” water, which is distilled at a central location and is dispensed through a tap near the sink. This is lab-grade water and can be used for making buffers, but not for cell-cultures or molecular biological purposes.
1. Autoclave most buffers, and undefined bacterial and yeast media
2. Don’t autoclave: corrosives (Acids, phenol, bases), solvents or volatiles (ethanol, methanol. Chloroform); liquids containing bleach, formalin, or glutaraldehyde; buffers with detergents, such as 10% SDS, since they can boil over; heat labile ingredients such as serum, vitamins, antibodies, and proteins; mammalian, plant and insect media; HEPES-containing solutions; DTT or B-ME containing solutions
3. Such un-autoclavable solutions must be either filter sterilized or autoclaved without the ingredient (which can be filtered and added later to the autoclaved material) (page 160)
1. Minimize junk around the working area
2. Wipe down the bench with antiseptic or cleaning agent before use
3. Bottles and tubes you are going to use should be sterile.
4. Set up the area to minimize hand movements
5. Have everything you need ready, and minimize moving away from the bench
6. Wear gloves, and change them frequently
7. Minimize exposure (keeping them open) of tubes, bottles. So work quickly when you open enzymes, primers, etc. Don’t leave a bottle open!!
8. Minimize fast motions in air, for it generates aerosols which can contaminate. So pipette gently, don’t wave pipettes with solutions in air, and don’t swirl the bottles.
9. Avoid pouring, and always use pipettes to dispense solutions (pouring generates aerosols)
10. Open all bottles & tubes at a 45o angle to minimize aerosols and contamination
11. Try not to put down caps, and if you need to put down, put it lid down
12. Have your own buffer stocks for restriction enzymes, PCRs etc. Don’t use the common stock.
13. Aliquot out everything, from primers to buffers to enzymes. In case of suspected contamination, you can always discard one tube and check.
14. Working with PCR: Have a separate area when you prepare PCR reactions, and a separate area (at least away from the PCR machine and away from places where PCR amplified tubes will be opened) for analyzing the amplified products. Briefly centrifuging the tube prior to opening will reduce contamination chances. Wear gloves and change them frequently. And finally, add DNA as the last thing to the PCR reaction
15. Working with RNA: Avoid using tubes and bottles that will touch RNA , and keep RNA buffers separate from other buffers, so that they won't be accidentally used and contaminated with RNAses.
16. During centrifugation: “pouring off” supernatants in the bench (do it in a hood), and opening centrifuged tubes without spinning them down.
17. Common mistakes
a. Pipetting too far up to the pipette
b. Touching the tip of the pipette against a bottle, the ground, the outside of a pipette container, or anywhere solid.
c. Using tubes which fell down.
d. Reusing pipettes while working
Everyone struggles with lab skills to begin with. I improved a lot after doing a studentship at the end of my second year and have become even more stringent from working in industry. You never stop learning. It takes practise and being taught how to exercise procedures properly, for eg - holding a pipette (something I was never taught at uni) and how to aim it into a well to prevent contamination.
Someone's lab skills depends on the experience they have. No one is competent with everything. Also people may have individual preference, such as forward or reserve pipetting.
It’s also important to make sure you understand all instructions before proceeding and research/use initiative if a technique isn't working.
Btw when I was doing my MSc, some postgrads didn’t have a clue when it came to basic lab techniques, including using pipettes, although that doesn’t mean it’s not advantageous to get a hang of these things before finishing a BSc.
Most important is to keep track of all records and be extremely organized, I use www.sparklix.com as my electronic lab notebook and it dramatically help me to be organize, find things easily and share data with other labs.
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