In the Genetics module of 7.02, you performed a transposon mutagenesis experiment using a mini-tn10 transposon to create an ara::lacZ fusion in KBS1 E. coli cells. You were able to phenotypically characterize your mutant as either "inducible" or "constitutive" by patching your mutants on LB/kanamycin/X-gal indicator plates in the presence or absence of arabinose. This allowed you to determine generally where your insertion might be - either under the control of the araBAD promoter (inducible with arabinose) or under the control of the araC promoter (which is constitutively on).

In the Recombinant DNA Methods module, you performed a PCR experiment in order to determine more precisely the location of the ara::lacZ fusion. In this tutorial, you will "sequence" your PCR product. Then, using BLAST, you will confirm the location of your ara::lacZ fusion more precisely.

In this tutorial you will do the following:

• Read DNA sequence
  1. View a DNA sequencing gel
  2. Read DNA from a trace sequencing file
• Use the BLAST program to:
  1. Submit your DNA sequence,
  2. Compare your DNA with other sequences in the databases,
  3. Determine the identities of your segments of DNA sequence,
  4. Learn more about the kind of information you can obtain about the function of a gene by knowing its nucleotide and protein sequence,
  5. Search the protein data-bases, and
• View the three-dimensional protein structure of part of your DNA sequence.