1. Log in Biology Workbench and click on session tools and start a new session. Name it tree or cladistics or some other easy to remember title. [ Start New Session ]

2. For this exercise - scroll down to tool bar/buttons and select "Protein Tools". There will be a scrolling text box in the page that comes up for you to choose an application. Select "Ndjinn" by clicking on it. A new page should open and you will need to enter information about the protein that you wish to find. It is capable of using Boolean operators (and, not, or), which we will use in future search lessons.

3. Type into the window "hemoglobin "(or some other protein) in the box and using the pull down to the right- choose "show all hits ."See note below on looking at sequence names.

4. In the colored windows below (these are various databases one could work with) check the box labeled - "SWISSPROT ."

5. Go back up and click ˇV "Search ."
Note: PIR , GENBANK and PDBFINDER may also be use for the initial searches as well. ( PIR1 contains validated sequences whereas others may contain partial or tentative sequences.) GENBANK might be useful to limit searches to a particular order of organisms, such as primates. Limitations of genbank include listing fragmentary portions of genes, which can be confusing to beginning users of the BW tool. PDBFINDER has the 3-D structures for the molecules, if that is desired.

A new page with another window appears with all the retrieved sequences (scroll down to the text box window). Scroll through the list of results and look at the organisms that have had that particular protein analyzed and submitted to the database. Highlight those in which you are interested. By using [ctrl] and clicking on an organism you can select several organisms to be compared.

Note: It can be hard to tell what some of the organism results show when using show all hits. If you check show 10 results per page, a checkbox list shows up ˇV uncheck those you do not wish to use from a page that has any you need then import the ones you wish to use from that page to your workbench. You will need to use the back page button on the browser (to return to the last searched page) and proceed to the next 10 hits page. Continue this process until you have all the ones you need. If there are none you desire then just go to the next page and ignore un-checking the choices. This is sometimes slower but provides more information at a glance.

Search Strategies and other notes:
a. Input a particular amino acid like myoglobin into BW and select view all sequences. If more than 100 show up you may wish to use a Boolean operator (AND, OR, NOT) to begin to limit the number of ˇ§hits"that you obtain.

b. Input a group of organisms such as whale or canidae and see what comes up.

c. You will need at least 3-4 different organisms showing up in a particular molecular analysis to be useful in generating phylogenic fragments to develop a composite tree. Make sure the same protein is used in clustered alignments. As an example, avoid mixing alpha and beta strands in these analyses.

d. There have been some problems in using sequences listed as ˇ§tentative"in the search results. Explorations involving these may provide bizarre results, but it may be an interesting sidelight to the activity to try to explain these results. Also, results labeled FRAGMENT may not be as useful unless the fragments are related portions of the molecule.

e. Molecules like prions can sometimes produce unusual results. There are a lot of mammals that can be studied, especially primates. In some cases these allow for very distinct evolutionary patterns.

f. Keep notes on the BW codes used for the species. This will make identifying the organisms on the different branches a little easier.

g. Use the view records button to find more information about the organisms. Their classification and other bits of information can be gleaned from this tool.