|
1. Start GCK and open the file construct#1 from the tutorial files folder provided.
2. You will mark restriction enzyme sites that are located exclusively in the polycloning region. Double-click on the polycloning segment which has a blue lined pattern and is located at about 3 o'clock.
|
|
Figure 8: Mark Restriction Sites
|
3. Choose Construct > Features > Mark Sites.... You will see Figure 8. Make sure that the commercial (no isoschiz) list shows in the popup menu at the top of the dialog. A list of enzymes will appear in the left scrollable area. Press the Add All -> button to add all the enzymes in this list to the right side list of Enzymes to mark.
4. In the middle section on the left, set the numbers to match that in the figure so you will be specifying enzymes that cut more than 0 times but less than 2 times -- i.e. unique cutters.
5. Finally, choose the middle radio button on the bottom left of the dialog box to specify that you want to see those enzymes which cut exclusively within the selected segment of DNA. Press the OK button to mark the sites.
6. You will see the construct labeled as shown in Figure 9A. Note that all the sites are crowded together. While all the newly marked sites are still selected choose Format > Site Markers > Automatic Arrangement to rearrange the site markers resulting in something that looks like Figure 9B.
7. When a site marker is selected, you have access to items in the Format menu to change the display of the site marker. Click on the BamHI marker and use the Format > menu to make the text bold and 10 point. You should end up with something that looks like Figure 9C.
|
|
Figure 10: Site Marker Get Info Dialog
|
8. While the BamHI site marker is still selected, choose Construct > Get Info.... This brings up Figure 10. If you wanted to, you could actually change the name of the enzyme (not a good idea) or enter comments in the comments text box. The horizontally scrollable area in the lower part of the dialog box indicates the actual sequence at that location. The dotted red rectangle indicates the recognition sequence and the two arrows indicate the actual cut sites. You can change the cut site by clicking on an arrow and dragging it. Press Cancel to close this dialog without saving any changes.
|
