Field of Science

Fear of cloning: time to stop stalling and just do it!

For several months I've been stalling on a relatively simple project whose completion would let us submit a nice paper.  This is the missing step in an undergraduate Honours student's project; I wrote about her project and what I need to do here.

  1. Starting with a circular plasmid containing the short 'Toxin' and 'Antitoxin' genes, I'll use inverse PCR to amplify a linear fragment that lacks most of the coding sequences of the Antitoxin gene.
  2. I'll also amplify (or find) a SpcR cassette.
  3. I'll ligate these two molecules together to create a circular plasmid with the SpcR cassette replacing the Antitoxin gene.  I could do this by blunt-end ligation (what the Honours student originally did) or do it as the student originally planned, using conventional ligation of 'sticky ends' generated by digesting both fragments with a restriction enzyme whose site is present at all the ends (she designed it into the primers).  I think she changed her plan because our stock of this enzyme was inactive.
  4. I'll use this plasmid (linearized by cutting somewhere in the vector) to transform the bacterium Actinobacillus pleuropneumoniae to SpcR.
  5. I'll use PCR of chromosomal DNA from the new resistant transformant to check that the original antitoxin gene has been replaced by the SpcR cassette.
  6. Next I'll do a transformation assay on this mutant to see if its competence has changed, with the wildtype and toxin mutant as positive controls.

Step 1 actually requires that I get off my butt and:

  1. Resuspend the primers at the appropriate concentration of TE (or water?).  (Check with the grad student.)
  2. Find the PCR reagents. (Ask the grad student).
  3. Find the template.  (Find info provided by Honours student before she left.)
  4. Learn how to run the PCR machine (Ask the grad student.)

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