The lab decathlon: Are you up for the challenge?
By: Roche Life Science
Posted: June 23, 2015 | Everyday Essentials for Research
While you might think the workings of a molecular biology laboratory are far removed from the performances of an athletic event, you are sorely mistaken. In fact, mastering the skills to succeed in the lab is not unlike those performed on the field -perhaps with much less sweat and physical injury, although this may be debatable for some.
Therefore, we have assembled the scientific decathlon: 10 essential laboratory techniques and tips that should be mastered in order to succeed on any molecular biology team:
1. Pouring SDS-PAGE gels: While pre-cast gels are readily available for purchase, being able to proficiently pour your own SDS-PAGE gels with consistency is a fundamental skill in any molecular lab. Indeed, appropriate acrylamide concentration is essential for optimal separation of your protein(s) of interest, as particular concentrations effectively separate proteins within a characteristic range. For instance, a typical 7 percent gel separates proteins between 40-200 kDa, while higher percentage gels (12-14 percent) are required for resolution of smaller polypeptides (<30 kDa). In SDS-PAGE, gel assembly is key - be sure your apparatus has no leaks (and check for leaks using distilled water), add APS and TEMED last (and only when you're ready for polymerization and to transfer your solution), be careful to avoid bubbles which can distort protein migration and always be sure to leave appropriate space for your stacking gel.
2. Pouring agar plates: A simple, but powerful microbiology method. While seemingly simple to prepare, there are some quick tips that can make this task even easier: Pour agar at about 50 degrees Celsius to minimize bubbles and to not solidify too quickly, gently pass the heat of a Bunsen burner flame to pop bubbles when pouring, let plates solidify and dry at room temperature until lids are moisture-free. Additionally, use color marking pens to quickly label stacks of plates (i.e. different antibiotics, nutrients) using vertical lines on the outside edges of stacks, store plates upside down in a refrigerator or cold room and be sure to examine plates for contamination prior to use.
3. Serial dilutions: Many common microbiological and biochemical techniques require dilutions of several orders of magnitude from 1:100 to 1:10,000 or more, necessitating the use of serial dilutions. While the advantage is greater accuracy of small volumes of dilution, consideration must be given to careful pipetting and calculation, as any error is propagated through subsequent steps. We recommend labeling all tubes at the start, changing pipette tips during each dilution to prevent contamination, mixing each sample well in between dilutions, and to keep track of finished steps by transferring tubes to a new row in the rack as you go.
4. Adjusting pH: This is essential for preparing your stock solutions. Accuracy is key: The basis of many protein biochemistry experiments relies on accurate pH of your solutions. Be sure your pH meter has been standardized and appropriately cleaned between uses. Also, when preparing a new solution, check for online recipes that may indicate how much adjustment needs to be made, as this can save loads of time dripping acids and bases into your solution at the pH meter.
5. Designing primers: Primer design software is readily available online and can save you tons of time and energy. However, it is essential to understand some of the fundamental basics of primer design for successful PCR, including optimal primer length (about 18-30 nucleotides) and melting temperatures (Tm), appropriate GC content, and avoidance of intra- or inter-primer homology that may result in self-dimers or primer-dimers.
6. Sequence alignment: Aligning sequences of DNA, RNA, or protein is an essential strategy for genetic and protein analysis. This can be performed on the small-scale (i.e. by hand) or large-scale, requiring sophisticated computational algorithms to analyze multiple, long sequences of data. There are numerous online tools and software available, including but not limited to, NCBI BLAST, ClustalW2 and T-coffee, among others. Become proficient at understanding complementarity, local versus global alignments, and the various available tools is key.
7. Multichannel pipetting: Notoriously tricky to master, but when done correctly, can save an enormous amount of time and energy. With the ease and availability of microplates for large-scale assays in modern labs, the need for multi-channel pipetting is increasing. In this case, practice is definitely important! Be sure your multi-channel pipette is correctly calibrated, that your tips are fixed and correctly set to the channels, and that the pipette is held at an even angle when dispensing and parallel to wells so tips never touch the well walls.
8. Differential centrifugation: The separation of cell lysates is a common strategy and understanding differential centrifugation and cell fractionation is a must. While low speed spins (400-500 x g) yield a pellet consisting largely of cell nuclei and unbroken cells, a moderately faster speed of 10,000-20,000 x g will pellet cytoplasmic structures such as mitochondria and lysosomes. However, further cell fractionation requires ultracentrifugation with rotors designed for high angular velocities that generate very high g forces. Indeed, a high-speed run at 80-100,000 x g will pellet microsomes, while 150,000 x g can bring down ribosomes.
9. Presenting your data: While the most important goal in your research is to produce good quality data, it is equally important to be able to effectively communicate your data to others at lab meetings, conferences and seminars. This takes thoughtful preparation, practice, and includes generating high-quality figures and graphs, clear descriptions of methods and experimental design, as well as thoughtful presentation of your findings using slides, posters or handouts.
10. Journal clubs: Besides being skillful at presenting your own data, you must master the ability to digest, analyze and present the relevant work of others. This requires learning background information and indications for the chosen study, understanding their methods, critically analyzing the reported data and interpreting findings independent of author conclusions. These strategies are essential for developing critical scientific thinking skills and for interpreting work from other labs.