Am I old-fashioned?

[Beatrice]

I *like* pouring my own protein gels. The PhD students were making fun of me today for not using the pre-cast gels. Oh, and for using wet blotting instead of the semi-dry apparatus for using film instead of the snazzy quant maching!

Am I over the hill?

[Nik Papageorgiou]

No, not really. But after pouring a million gels, I appreciate precasts. Same data, every time!

[hedge]

I like the precasts but I've found that the semidry isn't as efficient for transfer if you've got BIG proteins.

We don't have a snazzy machine but if we did, I'd miss the darkroom. It's just such a comforting ritual - and in the dark, no-one can see you cry over your bad results!

[Mad Dan Eccles]

I love(d) precasts, and wet blotting. *shrug*

[tideliar]

I love(d) precasts, and wet blotting. *shrug*

wot he said. I told my postdoc mentor that if he made my cast my own gels I wasn't coming to his lab. My project started out innocuously enough, but develoved (as I feared it might) into doing NOTHING but f*****g western's for 18 months.

3xday...
7 days/week
18 months

I hated it. Fucking technician with a PhD.

[Dr Mike]

I see nothing wrong with being old-fashioned. A lot of science is doing what you know, and sometimes it's easier to do what you know than take time to learn something ostensibly easier that will take time to get better at enough to see the difference. I tend to grab the innovations I see that are obviously very useful without being horrendously more expensive, but am wary of innovations that are really highway robbery while offering only a small shortcut in terms of ease/time. This leads me to having a weird grab bag of protocols old and new, all mixed together. I like to use calcium phosphate transfection because it's good enough for my cells and dirt cheap (am I the only one in the entire Universe still doing that??), compared to Fugene, say - but then I'm using a pulldown kit for my kinase assays because although expensive i see it as being massively easier and more reproducible than doing it ye olde fashioned way.

[tideliar]

Our gig in the lab, when something new came along, or a chance to try a new technique or a new bit of kit, was to do a simple cost-benefit ratio. My "salary" hours +old kit <= or => that $ of new kit/technique + "salary" hrs.

That's why I got my pre-cast gels. It was cheaper in the long run than being paid to pour thousands of gels (that's my excuse and I'm sticking to it!)

[challenge]

Ahh Bea, they laugh at me at my new job when I poured my gel instead of using the precasted ones. then they realised that they never use the 1% gels and that we don't have a precasted one... and then they saw how fast it was for me although, in general I like the pre cast ones since it makes it less messy (but if we don't have any, I can plan aroudnthe pouring the gel and keep myself occupied until it is ready to go).

I'm still stuck with the whole "if you know what the kit does, you can use it" but if you don't know Buffer A and buffer B, you need to learn... I was supposed to help trouble shooting the other week but got stuck with the first question "What did you do?" since the person started by saying "I added this buffer from the kit and then ran it".... not knowing really what or how it worked. That's when I feel ancient [but also knowledgable though, so I guess there is a positive side to it].

when working with bacteria I ended up mixing old and new protocols, for example there were this wonderful new tubes to "catch" chlorophorm and fenol when you spun them in a centrifuge. WONDERFUL. not only could I have larger quantities than 1.5 ml tubes with the c/f and therefore get less loss, I could minimise my own exposure to the smelly organics. They weren't too cheap though.... but boy, they sure made a place in my heart

[Freddie]

Yeah, I don't buy the "it saves time" argument. It takes, what, five minutes to assemble and pour a gel? Yes, it has to polymerise, but it's no big deal because you just do other things you have to do anyway while you're waiting. In my case I pour my gels and then use the 20-30 min pol. time to lyse and boil my samples. Gradients are nice when you have big and little proteins on the same gel, but they are really expensive and I don't like having to throw away all that Perspex every time, seems very environmentally unfriendly.

[Mad Dan Eccles]

For me it wasn't so much the time in pouring, as not the washing the damned plates and ensuring reproducibility. No bubbles FTW.

[Beatrice]

I never get bubbles in my home-made gels. Does anyone seriously think this is a problem?

Reproducibility in what way? As long as you have a ladder and compare your samples to a ladder does it matter if they don't migrate exactly the same way every time? At the end of the day you take your image and crop it around the relevant bands and you know they are right because you have controls etc. Just curious what sort of "irreproducibility" problem one could actually have. At least, I've never noticed that my experiments have problems and no boss or referee has ever questioned my bands.....??

[Mad Dan Eccles]

At the end of the day you take your image and crop it around the relevant bands

um, not if you're showing the gel and not just bands. I've done enough pull-downs, native band-shifts and Westerns not to want to risk having to re-run the damn thing because of a wonky comb or bubble in the stacker. Publication quality first time.

It's 'easier' if you're using one of the casters that let you only do two at a time, sure: but we had to use a 10-gel sandwich and that was a complete nightmare, until we switched to ready-made.

[Editor]

Well, I pour my own and I use the whole gel (because I typical inspect a high MW band and a loading control that's small) and I don't have problems getting a generally similar gel every time. But as I'm always saying, I'm anal-retentive when it comes to lab cooking so I am always hyper-precise.

Having said that, I'm training a tech tomorrow and I've decided to learn our pre-cast system so I don't teach her something that will be impossible to perform in a few years time when they finally stop making the Protean system! So I'm not a completely hopeless old fart-ess.

[Mad Dan Eccles]

I must admit, it's also very nice not to have to dive for cover because yet another lab mouse (not good enough to be a rat yet) has poured yet another quart of acrylamide all over the bench. bbbrrr.