feh

[opabinia]

I think I blew my Tubulin PCR. Looks like instead of using an internal standard, I grabbed a tube of the actual protein (a positive control, I assume) by mistake [Correction: That's cDNA, not protein. See comments.]. My gel has a pretty series of even bands all the way across. Just one band per lane, though, and of the exact size as the ones in the "Internal Standard Only" lanes. Crap. Where the Henley is the Tubulin internal standard, then? I looked everywhere.

Man, one crappy experiment can wreck your day.

I'm running an SR4 gel now. I don't think I can screw this one up.

***

I didn't know Po Bronson is such a hot shot. I guess I should come out from under my rock once in a while. This guy makes me feel like I've gone wide of whatever I should really be doing with myself. Graphic design? Acting? Writing? Queensland Lungfish breeding? I hope to learn someday, preferably soon. I mean, not that it took this book for me to realize I'm off on some kind of tangent, but some things look more obvious when they're printed in front of your face.

Comments

[spriggan.livejournal.com]

WTH???

[frederic.livejournal.com]

You're doing a pcr and talking about a tube of pure protein?

Screwed up banding (multiple bands of the wrong weight) is often due to doing the PCR anneal at too low of a temperature or having oligos that are nonspecific. Adding BSA, glycerol, or I think DMSO will also help with the specificity (or to make your polymerase happier). For BSA, I used to use 1 microliter of NEB's 100x BSA (from the restriction buffer kits) per 50 microliters of PCR mix. It made some stubborn rxns work (where it didn't in the absence of BSA) so I throw it in every time...

[lepidosiren.livejournal.com]

doing a pcr and talking about a tube of pure protein?

I was looking for a Tubulin internal standard (like tubulin, but smaller -- you know what I mean), and I apparently used a solution of the actual protein, probably meant for use as a positive control (as in, "Here's exactly where the band should be."). That's what I think happened.

My bands were single, clean, sharp, and specific. The problem is that there was only one per lane, and it was exactly where the "internal standard" band was in the no-cDNA-added, internal-standard-control lane.

The primers have been very good, and earlier PCRs with the anneal temp I used were also good. It really looks like I just used the wrong protein. Blegh.

[frederic.livejournal.com]

Does the solution of protein of any DNA that you can PCR off of? I'm rather confused.

Bands throughout often mean that one of your solutions is contaminated due to sloppy pipetting. Often happens when labs have common reagents for PCR and a few careless people...

[lepidosiren.livejournal.com]

Good God. Sorry about that. There's no protein anywhere. I'm just talking about cDNA. I used Tubulin cDNA, instead of smaller bits that are amplified by the same primers, to show whether the PCR reaction took place "evenly" in every micro tube.