File location		Maximum file size
Capturing images of results		Data Screen vs. Lab Bench
Keys to cases		Plasmid DNA
Program response time		Genomic DNA
Techniques used for case analysis		Troubleshooting

Location of files associated with cases: The DNA and protein sequences for the cases described here are located in the Cases folder that is included with the Case It download.  The necessary sequences for enzymes, probes, antibodies, or proteins for a particular case will be located in the same folder as the DNA sequences.  You can also use your own sequences to build your own case, or for other research purposes.

Capturing images of results: Ignore any comments in tutorial videos about using the ‘photo’ feature of Case It to capture images of gels, blots, etc. That feature is no longer supported, so you will need to capture images using screenshots.

Keys to cases: Contact mark.s.bergland@uwrf.edu for a link to the password-protected ‘keys to cases’ page, giving information on how cases were constructed along with screen shots of results for all case scenarios. Verified instructors only.

Techniques used for case analysis: Techniques used do not necessarily represent what would currently be done in a clinical laboratory, as DNA sequencing is increasingly being used for diagnosis. Our cases are intended to illustrate the variety of techniques that have been used to detect DNA and protein differences, and are designed to sharpen students’ analytical skills.

Program response time: In general, be patient and do not click repeatedly if the program does not immediately respond, especially when working with files larger than 500,000 characters in size*. It may take several seconds for the program to respond to commands if files larger than this are used, especially when selecting characters in text fields . Click and drag slowly when selecting characters under these situations. It may take a few seconds for the selection to be highlighted, so start and end the drag exactly where you want it to be, and after a few seconds the highlighting will appear. You can then right-click to copy it to your computer’s clipboard.

*Maximum file size: Case It v704 can display a maximum of about 2.67 million uppercase characters, each character representing a base pair or an amino acid. The largest files in the Case It folder are for the Salmonella case (4.6 million bp), so only the first 2.67 million of these bp can be displayed. Other cases have files considerably smaller than this (usually under 50,000 characters), so all bp are displayed.

Data Screen versus Lab Bench: The Data Screen (main screen) of the program should be used for case analysis and research purposes, as gels, blots etc. run relatively quickly and there are more features and options for DNA and protein analysis. The Lab Bench can be used to demonstrate how bands move across a gel over time, but it takes much longer to set up as it uses simulated equipment.

Plasmid DNA can only be run on the Data Screen (see below). Do not run plasmid DNA on the Lab Bench, as the open-circular and supercoiled configurations will not show properly.

If a DNA filename includes the word “plasmid”, and the file is not digested with a restriction enzyme, that file will be represented on a gel in both the open-circular and supercoiled positions, separated in the gel lane. The degree of separation is determined by numbers at the end of a file called OPTIONS.TXT, located in the Files folder of the Case It v704 folder. The default numbers are 1.15 for the supercoiled condition, and 0.85 for the open-circular condition. Changing these numbers will increase or decrease the degree of separation of the two configurations in the gel lane.

For a plasmid to be treated as “circular” by the program, three dots (periods) must appear at the beginning and the end of the DNA sequence (see the CRISPR exercise for an example). Note that there must be two identical sequences in the file, both with dots at the beginning and end. One of these will appear in the gel lane as in the open-circular position, and the other in the supercoiled position. Note: This only works on the Data Screen (main screen).

If cut at a single location, that DNA sequence will be linearized (represented by a single strand of DNA). For a plasmid to be linearized, no more than one restriction enzyme site can be present on the plasmid. After it is linearized, any number of restriction enzymes can be located and cut on the linearized plasmid sequence. This is just the way the program works – in reality, uncut plasmid DNA can have multiple restriction enzyme sites.

Genomic DNA: Files included with the cases are simple text files, most of which have a ‘.txt‘ suffix. Some of these text files have a ‘.gen‘ suffix, meaning that the files represent ‘genomic DNA’ (DNA from the entire set of chromosomes in a human cell). Since it is not possible to include the entire genome in these files*, the files contain only sequences that contain the mutation or markers for the mutation, and also sequences that represent the wild-type (‘normal’) situation.

When actual genomic DNA is run on a gel following digestion, a genomic smear results because so many bands are present. When a file with a .gen suffix is run on the simulated gel, a smear effect appears, but you can increase or decrease the transparency of the smear using a slider. This will reveal bands generated by the actual files in the case folders. Cases that use files with .gen suffixes rely on PCR or blotting to reveal the bands of interest.

Troubleshooting: If folders or files appear to be empty when you attempt to open sequences, it means the pathname to those files is too long because the Case It folder is nested inside other folders. Simply moving the Case It folder to your desktop will fix the issue. Similarly, if an error message appears when attempting to start Case It v7.0.4.exe for the first time (for example, “the file ‘HelpBioinformatics.txt’ is missing from the Case It folder”), simply move the Case It folder to your desktop, and restart Case It v7.0.4.exe. Make sure to leave the Case It v7.0.4.exe file inside the Case It folder at all times.