notethroughoutthetutorials,itemsrequiringactionsfrom.docVIP

NOTE: Throughout the tutorials, items requiring actions from you are denoted by and items which you should select or click on are bolded. Keep in mind before designing a specific primer or probe: You need to work with an updated database, and preferably the latest release of the SILVA database. You’ll need a computer with 4GB of memory. PROBE DESIGN Update your PT_Server using Probes | PT_Server Admin and select the name of your server. Select Build Server and DO IT at the warning message. Be patient for several minutes while your server builds (this will take hours for a large database). Choose one or several closely related sequences as your target sequence(s) for probe design. Only choose full length sequences. Mark the sequences by clicking on the mark icon and left clicking on the species Launch the probe design tool by selecting Probes | Design probes. Identify your PT-Server (probably user 1) Change ‘Min. group hits %’ to 100% to target all members of the group Keep in mind: if you have short sequences, increase the number beside ‘Max non group hits’ to allow for unmarked ‘short’ target group members which may be present in your dataset (none present for this exercise) Leave all other criteria as the default. Select GO. Your window should look like this: A status window will appear. Be patient, as the probe search may take several minutes. When the search is complete, a PD result window should appear. Below is an overview of the PD Result window: Target string: The sequence the probe is targeting. This is the reverse complement sequence of your probe. le: Length of the probe apos: absolute probe position, probes are grouped by letters (A, B, C, etc.) according to target site with the ‘best’ probes listed first. Overlapping probes are represented as + or -. The ‘best’ criteria takes into account the theoretical melting temperature and specificity. ecol: probe position relative to the E. coli alignment grps: number of sequ