So far you got acquainted with the plasmid pGEM-GOI and identified important elements within the plasmid essential for successful cloning.
The Promega site supplied you with information about restriction sites within pGEM. The introduction of GOI into pGEM changes this restriction map.  To get the new restriction map of the plasmid pGEM-GOI, you will use the WebCutter software. WebCutter produces restriction maps of given nucleotide sequences. We will examine a few of its features.   Software for sequence analysis (such as WebCutter) usually requires the sequence to be presented in FASTA format (this format is not numbered, and there are no spaces within it).
From now on you'll have to operate  three windows simultaneously.

To open a second window showing pGEM-GOI(reversed complementary strand) in fasta format click here.
To open a third window showing WebCutter click here.
Scroll down to the middle of the WebCutter page and follow the instructions:

  1. Enter a title for your sequence (pGEM-GOI).
  2. Copy the fasta format of the pGEM-GOI sequence from the second window.
  3. Paste this sequence in the appropriate box in WebCutter (in the third window).
  4. Select the type of analysis you would like (circular for pGEM-GOI since it is an intact plasmid).
  5. Indicate how would you like to display the restriction sites. First choose only : table of sites sorted alphabetically by enzyme name. Later examine other possibilities.
  6. Include enzymes cutting once in the display.
  7. Don't change the other defaults.
  8. Press the button Analyze Sequence at the bottom of the page.
If you need helpconsult the teacher

You received a long list of enzymes giving the following information: enzyme name, number of cuts (in this case only those enzymes cutting pGEM-GOI once), position of the restriction site and the recognition sequence (the information linked to more info is no longer available).

Does the enzyme BglI appear among the enzymes cutting pGEM-GOI once?
1. Yes
2. No