Monday, November 7, 2011
Where to go to troubleshoot protocols frequently used in the lab
Suggestions pooled from Erica Koval (Neurosciences Program), Peggy Ni (Immunology Program), and Elizabeth Todd (Department of Pediatrics Infectious Disease) at Washington University
Manuals from cloning kits often have a helpful Troubleshooting section (ie. Invitrogen's cloning kits) that includes explanations and solutions for various problems that might come up. For instance, I have definitely encountered issues with obtaining sufficient numbers of transformants and have tried some of the suggestions offered in manuals, such as altering the insert:vector ligation reaction.
Likewise, manuals for products used in qRT-PCR experiments from various companies (Applied Biosystems's TaqMan®, Invitrogen's SYBR® Green are popular examples) include "Good Laboratory Practices" sections that offer great PCR tips to prevent contamination and RNA-handling advice. Additionally, there are step-by-step protocols to set up standard curves and prepare the reactions that are incredibly detailed, often making it unnecessary to trouble-shoot.
Websites of companies that provide off-campus services, such as GENEWIZ, for DNA sequencing, have handy pages that provide tips on how to best prepare samples to submit, primer properties that yield good results, or suggestions to purify products.
2. Protein-centered experiments:
Of course, there is always word-of-mouth and getting helpful tips from either the senior members of the lab who have gone through the experience of troubleshooting a certain protocol or from other labs specializing in a certain troublesome technique. For example, I was experiencing difficulties with expressing sufficient levels of a protein; after asking a protein biochemistry lab for advice and hearing that adding a histidine tag increases production – most likely by targeting the protein to inclusion bodies more – I tried their approach and saw better results.
Performing Western blots is a fairly common lab protocol. For troubleshooting tips, the "Western Blot Analysis" and "Good Westerns Gone Bad" sections in the manual for the Odyssey® imaging system has some helpful advice, including suggestions on different blocking buffers, concentrations of various detergents to use, etc. Another website to look at is the Cell Signaling Technology® FAQs section (under the "Support" heading). Here, you can find various things to try if a certain antibody isn't working or ways to ensure reproducible Western blot results.
3. Cells and tissues:
The Current Protocols in Immunology book, published by John Wiley & Sons, Inc., offers protocols on a variety of topics, with scientists renowned in a certain field contributing the information on that particular topic. A solid source of information, it is great for learning the background of a certain experiment as well as for finding tips or alternate protocols. Growing finicky cell lines, for instance, is something detailed in the book that was very helpful for members of my lab who were unfamiliar with the specifics. Additionally, the Current Protocols series includes topics such as molecular biology that are applicable and useful across many different fields.
University core facilities offer information on their websites for flow cytometry, a ubiquitous technique in immunology labs. Washington University, Yale, and UC Berkeley are just a few examples of places to find recommendations on antibody staining to yield better cell sorting results, eliminate overcompensation to analyze the data more accurately, etc.
There are multiple places to find good protocols or troubleshooting advice for immunohistochemistry. One is the Cell Signaling Technology® website. Under the "Support" heading, the Protocols section offers detailed instructions for both frozen and paraffin samples, with buffer components included for different antibody diluents and antigen unmasking procedures. Another way is to go to the antibody company website and look up recommended protocols. Abcam®, for instance, has a lot of information and suggestions. Various antibodies include reviews and ratings submitted by scientists as well as questions others have asked that have been answered by the scientific support team. Additionally, the website provides troubleshooting tips for immunohistochemistry, with advice for addressing issues such as high background or non-specific staining.