Mystery...Solved

After several tedious weeks of subcloning from a phage clone, several discouraging months of creating a NotI site by mutagenesis, a couple of boring weeks of inserting a GFP cassettee into the created NotI site, a few tiresome days of subcloning it into an injection plasmid pW8, an intense week of injections, 3 anxious weeks of waiting and praying, 5 hurried minutes of dissecting and preparing slides of different fly tissues, finally, the expression pattern of GFP tagged k5, a protein expressed ubiquitously, is ready to be examined.

(For those who don't read Biology, this means, I had a hard time making this thing. It's finally done. I am going to check the slides under the microscope, and I am expecting to see bright green, green, green color everywhere.)

This is the moment of truth. Relax. Breathe in. Breathe out.

I put on a slide. It is a female ovary. I look into the eye pieces of the fluorescence microscope. I see--darkness, darkness, darkness everywhere.

I am supposed to see green! Where the hell is the green? What's with all this darkness?

I put on another slide. It is some other tissue I dissected out from a female fly. I don't even know its name. Look, and see darkness.

Another slide--darkness. On all slides I see darkness, except the one with male testes on it. On that one I see bright green light along the sperm bundles, which looks exactly like another GFP tagged protein I have made and examined a few weeks before. That protein, k5t, is a testis-specific protein and is supposed to be seen only in testes.

After dissecting and examining some more flies, both of k5 and k5t, I reach a conclusion: they look the same, which, I can't say is impossible, but is very very improbable.

What's happening?

The only logic explanation I can come up with is: I pulled out the wrong tube of DNA to do the injection, so these alleged "k5-GFP" flies are in fact "k5t-GFP" flies.

The result of a subsequent PCR test is consistant with this explanation.

(For those who don't read Biology, this means: I am screwed.)

Oh, what a stupid mistake! What a terrible waste! But what can you do now? Punish the flies? Abuse them? Or raise the "lab-soberness alert" to orange?

I will begin to do some injections tomorrow, this time, with the right DNA.