trinityvixen (![[personal profile]](https://www.dreamwidth.org/img/silk/identity/user.png)
trinityvixen) wrote2004-07-27 03:47 pm
trinityvixen) wrote2004-07-27 03:47 pm
Is it so wrong?
Yesterday, I didn't install stuff on my boss' new computer. I didn't do a Western blot prep though I had the time. There's just going to be a lot of time in the next few weeks in which to do stuff. So, I procrastinated yesterday a bit. But today I resolved to work work work, and got right on it.
Well, tried to, anyhow. I feel like a total failure. The first thing I learned to do here was make these acrylamide gels. I spent the better part of half an hour trying to figure out how the mini-gel plates snapped into the frame. I tried looking at the manual online because ours here is AWOL, but the manual is for something we don't have, ours not being sold anymore, I gather. Half an hour to figure out how to put tab A into slot B and snap the thing in place. Jesus. Columbia education, and I can't put 2 and 2 together.
Eventually, when the frame was situated so that it was tightly clamped in place, I started to make the recipe for the separating gel. I mixed and poured, poured and mixed, then quickly droppered in the gel. I added dH20 to it, too, so it wouldn't dry out. There were a few too many bubbles, but the water got most of that out. The problem was not getting things out, however, it was keeping things in: my gel started to leak. Both gels, as it turned out. Sigh. It's a 1.5 hour process to run the gel, then a 2 hour one to transfer it to nitrocellulose paper, and there would be no time to do it unless I wanted to be here until 8-9pm. Because I didn't have the samples ready yet (I was going to let the gel set and then go make the samples), I gave up. I also nearly had a heart attack over thinking I didn't know how to make the gel any more. It took 30-40 mins to set, and I don't remember the big gels ever taking that long. The left over gel did eventually solidify in the 50 mL tube, but still, should it have taken that long?
After doing something else, I decided it would be best to try out the appartus with just water and see if I could figure out how to keep it from leaking. Lost another 10-20 minutes on that. Currently, it looks like it may still be holding, but I can't figure out why this slight rearrangement doesn't leak and the thirty-odd variations on the theme didn't (it's not like I have much room or adjustment to play with here). Worse, I can't leave this set up like this and just drain it and fill with gel because it's got to be clean before loading gel into it. So, I'll have to take it apart, wash it, and hope I remember what infinitessimally small thing is different about it now.
Le sigh. I feel like a failure.
Well, tried to, anyhow. I feel like a total failure. The first thing I learned to do here was make these acrylamide gels. I spent the better part of half an hour trying to figure out how the mini-gel plates snapped into the frame. I tried looking at the manual online because ours here is AWOL, but the manual is for something we don't have, ours not being sold anymore, I gather. Half an hour to figure out how to put tab A into slot B and snap the thing in place. Jesus. Columbia education, and I can't put 2 and 2 together.
Eventually, when the frame was situated so that it was tightly clamped in place, I started to make the recipe for the separating gel. I mixed and poured, poured and mixed, then quickly droppered in the gel. I added dH20 to it, too, so it wouldn't dry out. There were a few too many bubbles, but the water got most of that out. The problem was not getting things out, however, it was keeping things in: my gel started to leak. Both gels, as it turned out. Sigh. It's a 1.5 hour process to run the gel, then a 2 hour one to transfer it to nitrocellulose paper, and there would be no time to do it unless I wanted to be here until 8-9pm. Because I didn't have the samples ready yet (I was going to let the gel set and then go make the samples), I gave up. I also nearly had a heart attack over thinking I didn't know how to make the gel any more. It took 30-40 mins to set, and I don't remember the big gels ever taking that long. The left over gel did eventually solidify in the 50 mL tube, but still, should it have taken that long?
After doing something else, I decided it would be best to try out the appartus with just water and see if I could figure out how to keep it from leaking. Lost another 10-20 minutes on that. Currently, it looks like it may still be holding, but I can't figure out why this slight rearrangement doesn't leak and the thirty-odd variations on the theme didn't (it's not like I have much room or adjustment to play with here). Worse, I can't leave this set up like this and just drain it and fill with gel because it's got to be clean before loading gel into it. So, I'll have to take it apart, wash it, and hope I remember what infinitessimally small thing is different about it now.
Le sigh. I feel like a failure.
no subject
"I am Godlike. Except that He's perfect, and I fuck up all the time. That's the only difference, really."
(My friend
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*hugs* you are SO not a failure! -- of course it takes a while to settle into a new job, and at least you were lucky enough to get one straight out of uni, which is more than I can hope for ;) ..... *sigh* the woes of an arts student......
Some words of wisdom
First of all are you using the old-fashioned apparatus or the new BioRad model with the green edges?
To check for leaks without having to reassemble the apparatus: instead of using water, use 95%-100% ethanol. It will evaporate while you are mixing the gels, and the plates won't be further contaminated.
To stop leaking while it happens: if you are using the old-fashioned apparatus, there is that ridge of plastic that you snap the plate holder under. If it isn't tight enough, or if the base of the plate holder is not flush against the rubber gasket, you will get leaking. You can prevent it by checking to see if the space holders are aligned properly (number one cause of leaks). If it leaks anyway, push down at the top of the holder immediately, and stuff a wad of KimWipe between the ridge and the top of the holder. The pressure will halt the leaking.
Try using butanol or isopropanol to keep the gel from drying out instead of water. It will not evaporate away even if it sits for over an hour, and you can pour stacking gel on top of it - the alcohol will displace to the top.
Yes, it CAN take that long for the gel to polymerize, especially the stacking gel since it has less acrylamide. Sometimes slow polymerizing is due to the quality of the APS, which should be remade fresh every week or so. You can check if it polymerizing decently by looking at your tube of leftover and checking for bubbles. If you see little bubbles floating around, that's polyacrylamide, then you're good.
You can flush out unpolymerized solution, debris, etc with running buffer/
I have broken so many glass plates for Western blots! So annoying. Lately my blots have not been working - I get very poor signal when I develop the membranes. I don't know if it is too to low protein concentration or if one of the antibodies decided to suck. They worked a month ago ... blarg. Western blots are a two-day process, even if you stay till 9pm, and I hate when they don't work right. Ah well, 2-D PAGEs are worse!
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(Anonymous) 2004-07-27 07:21 pm (UTC)(link)Liz M
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GNK