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The waiting game begins again...
I've been trying to TAP-tag my favorite protein for the last five weeks (basically, this just means inserting a nucleotide sequence at the end of the gene so it will be translated with the protein) so that I can do various TAP-taggy sorts of things. Yeast is a great model organism for things like transformations--all I have to do is PCR amplify the TAP tag using primers with ends that are homologous to the end of my gene, then put the PCR product directly into yeast (no cloning, no vector, nothing), and the yeast will use homologous recombination to transform itself. I did the transformation several weeks ago, and have been trying for at least the last month to verify the tag by Western blot (this involves breaking open a bunch of yeast and extracting their proteins, then probing for the tag with an antibody). Strangely, my Westerns have been completely blank. Not faint, blank. No smudges, nothing. I got several different positive controls from labmates, and have been able to get those to show up perfectly, but still saw nothing in my sample lanes.
Yesterday I finally checked over my primers. Sure enough... not only did I leave the STOP codon in the sequence, I also took the wrong sequence from the end of the gene--instead of the last 50 nucleotides, I used the second to last 50 nucleotides. So my primers were both in the wrong place AND contained a STOP codon before the tag, so of course I never saw anything on the blot--the TAP tag never got translated.
Alas.
I ordered new primers and they should be here later this week. Meanwhile, I just have to sit and wait again.
At least now I know it's not my bench skills that are bad. Messing up primers is much, much better than being incapable of doing a proper Western.
While I'm waiting for primers, though, I get to pack. We move tomorrow and Friday! This weekend I'm going to head to IKEA and get lots of fun furniture-y things, like a dresser, and some more chairs, and maybe a rug or two (okay, rugs aren't furniture). I'm so excited!
I've been trying to TAP-tag my favorite protein for the last five weeks (basically, this just means inserting a nucleotide sequence at the end of the gene so it will be translated with the protein) so that I can do various TAP-taggy sorts of things. Yeast is a great model organism for things like transformations--all I have to do is PCR amplify the TAP tag using primers with ends that are homologous to the end of my gene, then put the PCR product directly into yeast (no cloning, no vector, nothing), and the yeast will use homologous recombination to transform itself. I did the transformation several weeks ago, and have been trying for at least the last month to verify the tag by Western blot (this involves breaking open a bunch of yeast and extracting their proteins, then probing for the tag with an antibody). Strangely, my Westerns have been completely blank. Not faint, blank. No smudges, nothing. I got several different positive controls from labmates, and have been able to get those to show up perfectly, but still saw nothing in my sample lanes.
Yesterday I finally checked over my primers. Sure enough... not only did I leave the STOP codon in the sequence, I also took the wrong sequence from the end of the gene--instead of the last 50 nucleotides, I used the second to last 50 nucleotides. So my primers were both in the wrong place AND contained a STOP codon before the tag, so of course I never saw anything on the blot--the TAP tag never got translated.
Alas.
I ordered new primers and they should be here later this week. Meanwhile, I just have to sit and wait again.
At least now I know it's not my bench skills that are bad. Messing up primers is much, much better than being incapable of doing a proper Western.
While I'm waiting for primers, though, I get to pack. We move tomorrow and Friday! This weekend I'm going to head to IKEA and get lots of fun furniture-y things, like a dresser, and some more chairs, and maybe a rug or two (okay, rugs aren't furniture). I'm so excited!
