Matt Gethers/CRI, Thailand/Labwork/Generating the HmgR Mutant/Week of 6.8.08

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Amplification of Homologous regions

We're trying to make knockouts of both HmgA and HmgR. Do make the knockouts, we're going to use the cre-lox system. As such, we need the sequences up and downstream of each gene. These upstream and downstream sequences will then be cloned into a vector including an antibiotic resistance marker and flanking loxP sites.

Sopapan (spelling?) has already amplified the sequences up and downstream of HmgR (she calls them 3 and 4 respectively) as well as the region upstream of HmgA (she calls this one 2). She had trouble amplifying region 1 (downstream of HmgA), but she realized that she had used the wrong primers. I need to use primers 2724 and 1033 and then digest the amplicon with NcoI. The resulting fragment will be what I need.

Today I'll gather all the reagents I'll need and put together the PCR protocol.


Today I'll amplify the ~2.5 kb fragment from the pareu genome, digest it with NcoI, run the resulting digestion product out on a gel (1% Agarose), and extract the band from the agarose.

I also started a culture of of Pseudomonas aerugenosa (henceforth Paeru) at 10:15 AM in ~5 mm LB to make a glycerol later today.

I need to check if/decide:

  • Do they do a PCR clean up before digestion?
    • No
  • They usually determine the concentration of PCR product before digestion and if so how (nanodrop?).
    • No.
  • Should I digest all the product or save some?
    • Digest 10 μl of product.
  • Figure out how to use the gel imager.
  • Where do I incubate at 37oC?
    • There's an incubator right outside of my lab area.

It looks like the PCR failed (and the digest following it), so I'm going to repeat both. This time, however, I'm including a positive control, a reaction containing 1032 and 1033 primer mix. I'll use the same protocol as before, except I'll add a 7 minute 72oC final extension step at the end.