You will now have the opportunity to "call" your own sequences. In the frame at the right, you will again see Segments 1, 2, and 3, with the bases from the ABI trace. Scroll down and you will see the trace diagrams for Segments 4 and 5. Note that the nucleotide positions and bases have been deleted so that you can call the bases.

A few tips for base calling:

• The periodicity of bases over short stretches (such as the segments) should be reasonably constant. In other words, bases should occur at evenly spaced intervals.

• G is generally weak, and for some reason, a G following an A often shows a markedly decreased signal. Note, for example, bases 57 and 137 in segments 1 and 2, respectively.

Enter the sequences into the text boxes above each trace (labeled Segment 4 and Segment 5). You may find it useful to use the mouse arrow to keep track of where you are on the trace diagram while you are typing in the sequence.

Now answer Question # 1 of the handout: What is the sequence of Segment 4?

When you are confident that you have entered the correct bases, scroll to the bottom of the screen and click the button labeled "Assemble Total Sequence." You will see the five segments joined together in the window underneath the button.

Copy the sequence: Highlight the sequence with your mouse by clicking on the first nucleotide, and then dragging the mouse across the sequence until the end. Use the "copy" command (Edit -> Copy, or Ctrl-C [PC] / Command-V [Mac]). If you are using an Athena workstation, the middle mouse button will copy the text after it has been highlighted.

At this point, you may also want to save your sequence in a separate file on your computer in the event that you would like to come back with this tutorial without having to re-read the sequences.

Part I Previous:
B. Reading a DNA Sequence

Part II Next:
Using the BLAST Database