Bloody agrobacterium!
Aug. 19th, 2002 06:25 pm![[personal profile]](https://www.dreamwidth.org/img/silk/identity/user.png){kind=small}
hnpccI am going nuts trying to purify a plasmid back out of Agrobacterium.
The problems seem to be many.
1. It's a low copy number plasmid. So there's not much of it to start with.
2. Agrobacterium stinks. Really, really stinks. You thought E.coli was bad - this is worse. And the smell lingers. I can smell the smell in the DNA I've allegedly purified. I am now known as the "smelly bacteria" person in the lab.
3. It grows slowly. This means thinking at least 2 days ahead. Sometimes this doesn't work. Weekends particularly get in the way of my thinking.
4. So far I've tried 3 methods of plasmid purification. None have worked. When I say worked I mean I get DNA but it won't digest with anything. Argh. I am trying very, very hard to avoid doing a total DNA and a southern. Why? Cos it's a hell of a lot of work to confirm an insert, which really should not take that much work at all.
5. Rifampicin makes everything go orange. The bacteria is resistant to rifampicin. By the end of this so will I be - not least cos it's dissolved in DMSO, nicely helping anyantibiotic that gets on to my skin to enter my bloodstream.
6. Did I mention the smell? Honest, it's bad.
7. PCR is doing "funny things". Just to add to my woes. OK, strictly speaking this is not the agrobacterium's fault, but it's not helping confirm my inserts. One is fine. One is not coming up at all, and one has - for no reason that I can see - gained an extra 1-200bp. I'm back on the "aliens are messing with my DNA" theory that worked so well during my post-grad.
8. It is worrying me slightly (OK a lot) that the post-doc I am working with and who is a plant person is also stumped. And - to quote - "this should be the easy bit. You just get the plasmid back out, confirm it and transform. We've never had a problem at this stage before". Oh great, I am the Anti-Bob. Can I break it? Yes, I can.
9. It sometimes inserts bits into its own DNA. I have no idea how frequently this happens. It's not a prospect to make me happy.
10. Even when I transform the plasmid DNA back into E.coli I can't get the plasmids to digest. I'm blaming the agro - and changing all my solutions. *sigh*
11. Triparental mating. Can anybody tell me what, exactly, is going on with this and why the PRK2013 takes my plasmid too?
I have so seriously never had this many problems confirming a friggin' insert. Part of the problem seems likely to be that we are using a different strain of agro to the one that the post-doc was using previously. None of us have used this before (well I certainly haven't - transforming plants didn't really come into cancer research or throwing parcels for some reason). Part of it is almost certainly something to do with my solutions. Which means heaps of solution making tomorrow. Argh.
Still, I'm not working nights and I'm not throwing parcels. And I'm enjoying myself. I just want this bloody insert to show it's head so I can transform my lettuce and tobacco.
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The problems seem to be many.
1. It's a low copy number plasmid. So there's not much of it to start with.
2. Agrobacterium stinks. Really, really stinks. You thought E.coli was bad - this is worse. And the smell lingers. I can smell the smell in the DNA I've allegedly purified. I am now known as the "smelly bacteria" person in the lab.
3. It grows slowly. This means thinking at least 2 days ahead. Sometimes this doesn't work. Weekends particularly get in the way of my thinking.
4. So far I've tried 3 methods of plasmid purification. None have worked. When I say worked I mean I get DNA but it won't digest with anything. Argh. I am trying very, very hard to avoid doing a total DNA and a southern. Why? Cos it's a hell of a lot of work to confirm an insert, which really should not take that much work at all.
5. Rifampicin makes everything go orange. The bacteria is resistant to rifampicin. By the end of this so will I be - not least cos it's dissolved in DMSO, nicely helping anyantibiotic that gets on to my skin to enter my bloodstream.
6. Did I mention the smell? Honest, it's bad.
7. PCR is doing "funny things". Just to add to my woes. OK, strictly speaking this is not the agrobacterium's fault, but it's not helping confirm my inserts. One is fine. One is not coming up at all, and one has - for no reason that I can see - gained an extra 1-200bp. I'm back on the "aliens are messing with my DNA" theory that worked so well during my post-grad.
8. It is worrying me slightly (OK a lot) that the post-doc I am working with and who is a plant person is also stumped. And - to quote - "this should be the easy bit. You just get the plasmid back out, confirm it and transform. We've never had a problem at this stage before". Oh great, I am the Anti-Bob. Can I break it? Yes, I can.
9. It sometimes inserts bits into its own DNA. I have no idea how frequently this happens. It's not a prospect to make me happy.
10. Even when I transform the plasmid DNA back into E.coli I can't get the plasmids to digest. I'm blaming the agro - and changing all my solutions. *sigh*
11. Triparental mating. Can anybody tell me what, exactly, is going on with this and why the PRK2013 takes my plasmid too?
I have so seriously never had this many problems confirming a friggin' insert. Part of the problem seems likely to be that we are using a different strain of agro to the one that the post-doc was using previously. None of us have used this before (well I certainly haven't - transforming plants didn't really come into cancer research or throwing parcels for some reason). Part of it is almost certainly something to do with my solutions. Which means heaps of solution making tomorrow. Argh.
Still, I'm not working nights and I'm not throwing parcels. And I'm enjoying myself. I just want this bloody insert to show it's head so I can transform my lettuce and tobacco.
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no subject
Date: 2002-08-19 01:48 am (UTC)Not only is this entirely beyond me, it contains strange references to things like "transforming tobacco". It's like it fell out of an SF script or something. And then, nestled among all the stuff that makes me go "wow" is the bit that makes me laugh out loud:
"I am the Anti-Bob. Can I break it? Yes, I can."
I think I might use that as a sig file sometime.
no subject
Date: 2002-08-19 02:07 am (UTC)Anyway, I tell you what does stink. There is a lab around the corner that is using Deinococcus radiodurans. That really stinks.
no subject
Date: 2002-08-19 09:32 am (UTC)don't understand any of that, sounds very impressive and makes my only glimmer of 'ooh i wonder' seem very humble.
We had a meningitis [sp?] outbreak some years ago and everyone was jabbed and had to take an antibiotic that made you orange, [and everything that came out of you was orange too.] scary stuff.
hope you're no longer anti-bob and having some success.