Cloning is the fucking bane of my existence.
Seriously. I do not understand how something so simple and straightforward as cut-and-paste could thwart me thoroughly at every turn. It should have taken me seven days tops to generate this construct. We’re going on fourteen now. Part of the delay is that nobody bothered to reorder the appropriate antibiotic when it got used up, but most of it is just that shit isn’t working, and since there’s no good reason it shouldn’t work, I must be fucking something up. Except I don’t know what I’m fucking up because this makes no goddamn sense.
It’s not that I don’t have any experience with this. It’s not that I don’t understand it. I plan very carefully and do everything “right” and then get a fucking incomprehensible product (or none at all). This is child’s play and I’m a motherfucking postdoc. I should be able to do this shit in my sleep.
I mean really, I cloned this fucking fragment into the vector with the same fucking sites so that I could send it for sequencing, confirmed the insert by blue/white screening AND colony PCR and now the same fucking enzymes won’t release it. What the fucking fuck?
This is not difficult. I routinely do six more difficult things than this before breakfast. So how is it that I suck so damn hard at this?
I want this done so I can do some actual science. That’s why I’m here, right? Right?
Weeks like these I want to hang up my lab coat.
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Cloning 1 part science, 3 parts voodoo magic. Immunofluorescence is very similar except it's more 1 part science, 5 parts dark arts. If you start finding mysterious tatoos on your arm, run away!
Which is to say: This shit happens. It's such a pain in the ass and a waste of time. I'm kind of happy my PI outsources cloning to a company so I don't have to fuck with it anymore.
Word to biochembelle. I've had standard shit blow up in my face and hoodoo 'not even 7th year postdoc can do this shit' turn out beautifully. There are many assays I never figured out why they started working, I took that shit and ran with it.
Of course, the entire time up until it works you wonder if half your brain feel out someplace cause the shit makes no sense.
Yep. You peeps know what's up.
Methylation?
I traded part of my cloning with a lab mate when it didn't work... they did one part of the whoel line since I couldn't seem to get colonies on my plates. (this time, last 4 times it worked great)then I fianlly got some colonies but lots of empty vector still glowing nicly... science huh! such a fickle lover.
If all else fails (have good DNA first) then just sacrifice that odd plate with the bunsen burner and invoke magical sciency gods and it should solve itself. In time. right?!
and I'm sorry about all the spelling errors. I'll blame it on the hoodoo.... sorry.
Shouldn't be methylation. One enzyme has no methylation sensitivity, the other is sensitive to CpG methylation in mammalian gDNA, but since these are plasmids it seems a moot point. Unless I'm missing something?
Chall - I would LOVE to trade this out to a lab mate. Unfortunately, I am the most experienced cloner here. :(
E. coli has methylases that block some restriction enzymes, depending on the flanking sequence of the restriction site. Xba I is a sensitive to this methylation.
Anyway, subcloning hell suckes asse. I once dicked around for fucken *months* trying to build a fucken construct.
You're talking about dam and dcm methylation, right? The enzymes I'm using aren't sensitive, and the strain is dcm- anyway. Can't find any info on whether it's dam- so I assume it isn't. I'm using NcoI and NotI, both of which should be OK in either case.
I'm throwing in the towel on this for today since I'm all out of ideas. Hopefully, getting sequence back tomorrow will be enlightening.
I feel like every damn time I embark on a simple cloning strategy I end up dicking around for fucken months. Really wreaks havoc on my sense of self-worth. Glad to know it's not just me though.
AA> The one who I traded with had no "real" experience... I just needed someone else to try cleaning up and adding AAAs to try and make it work. Desperation and shooting in the dark! ;)
I don't know which vector you're using but I remember from my old cloning - not the one I'm using right now - that XhoI was a better enzyme for me than the NotI for the vector used (had a bunch of them in there... still have hard feelings against NotI and NheI; probably not 'their' fault but it never really made a good thing.
The other thing, which I guess you have doublechecked but still; you're sure you don't have any sites for those enzymes in the sequence you want to clone? (just double checking)
Simplest explanation? some jackhat left the enzymes out. Or the power went out and nobody told you.
unless you're running a successful positive control?
Anyway, cloning can be a royal pain.
I once wasted two weeks with a VERY impatient PI breathing down my neck because my primers were *put in the wrong frickin tubes*. They were ordered correctly, even synthesized correctly, but then the core facility put them in the wrong. frickin. tubes. When we figured it out- after mass specing them- the core people asked whether *I* mixed up the tubes. I felt like slapping them silly. It was NOT my best moment.
Just did that with my westerns. Two flipping weeks of mostly 10 hour days. Oh my agony.
As for cloning, most of mine was rather cooperative, except for the tagged plasmid. I was new and a PI & her post-doc from another lab were teaching me how to clone & do some SDM. The wt plasmid grew fine but the tagged one wouldn't grow at all. No colonies. Turned out that was a Kan resistant clone, not Amp. Hence, it wouldn't grow on an Amp plate, despite this PI telling me to use Amp plates. :)
Good to know this sort of chaos will continue to happen when I'm a post-doc. I was beginning to wonder whether I deserved to graduate when I couldn't get this stupid western to work... =/
Turned out that was a Kan resistant clone, not Amp.
HAhAHAHAH! I totally did this once, too!
I totally despise subcloning. Had a bad experience in grad school. To this day I hate subcloning, even when it works.
If I had a blog, this is the entry I would have posted today.
Yeah I spent 3 months trying to clone an shRNA once. It was not pretty (the mouse one cloned fine; the homologous human one decided to be a douche). Cloning is the most complex straightforward procedure I've ever encountered (A close second: PCR).
You can do diagnostic single cuts to see if either enzyme alone is capable of linearizing the vector; that will tell you if one isn't working (but not why). The most probable explanation if one doesn't cut is that the enzyme has kicked the bucket. If it's not that, the enzyme site might have mutated (it's rare, but I have seen it happen, which is why I am now anal about sequencing all my cloning).
turn and around 3x times, spit in the sink and make sure the moon is in the "right" spot. That is what I was told at least about cloning....
Bah! I just got sequence back on the fucker and whatever the fuck is inserted in there has zero alignment with the sequence that *should* be in there. This doesn't tell me shit about what went wrong, other than I seem to have spurious priming (which results in the exact same size product as the one I actually want) when generating the insert via PCR cloning.
Which of course still doesn't explain why enzymes won't release the insert - regardless of the sequence, I did clone it into these same sites.
Fuck this shit.
I'm developing a new strategy.
Cloning is the fucking devil.
second word to biochembelle. i've effing hate cloning. i'd love to pay a minion (i mean) tech to make clones for me! good luck!! last time i had issues, my primers were for the wrong gene.
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