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DNA Strider Manual: Bio312 2000
Bonnie K. Baxter, Lewis & Clark College

Your recombinant plasmid is a combination of two sequences: your pCITE vector and your PCR fragment. Files for both of these sequences have been provided. To merge them and create a new sequence, first open the sequence file for your PCR product.

Within this sequence, find the restriction sites that you used to clone your fragment. You can do this "by eye" if you like, particularly since you know where these sequences are. Your other options include creating a restriction map or report (from the enz menu) or using the Find command (from the find menu, or command-F) to search for the sequence of your site. If you do not remember the recognition site sequence for a given enzyme, you can find this using the "enzymes data" command under the enz menu.

Repeat this process to find the relevant restriction sites in your vector sequence. Now use the "copy" and "paste" commands to create a new sequence. Select the sequence of your PCR product that was cloned into your vector (and only the sequence that was cloned in). Choose "copy" under the edit menu (or command-C). Now move to your vector sequence and select the region that was removed in your cloning. Select "paste" from the edit menu (or command-V) to replace this sequence with that of your PCR product.

NOTE: be sure that you create a sequence that accurately shows your fusion points. Each relevant restriction site should be included in full, but only once. Don't create an NcoI-NcoI duplication, for example.

Saving your recombinant sequence

Now that you have a new, recombinant sequence file, save it. BE SURE, however, NOT to replace the vector sequence file with your new sequence. (i.e. DON'T choose "save"--choose "save as" instead.) Give your file a name that reflects the name of your new plasmid--something like pCITE2-LTV1-HisTag, for example. Save it to the "Bio312 sequences" file if you do not have a disk on hand. Otherwise, save it to your own disk. (Sequences saved to the hard drive may be deleted without warning, so make your own copy ASAP.)

Creating your map

Follow the steps outlined in step 2 for the vector plasmid to create a new graphic map of your recombinant plasmid. Include all enzymes that cut 1, 2, or 3 times.

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