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Cloning: Randomly recombined Plasmids?


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6 replies to this topic

#1 astartus

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Posted 13 July 2010 - 12:45 AM

Hello everybody.

It seems I have to do a very, very hard cloning.

In theory it's very easy: I just want to cut a lentiviral plasmid with XbaI, blunt it and religate the plasmid just to kill the XbaI side. OK ... after the transformation I get colonies. But as soon as I do Miniprep, I see that all the plasmids are different and have not the size of original plasmid anymore.

I wanted to ask, if anybody has any experience with mysterious things like that? I used another lentiviral vector (which has the same basic elements, but only other markes) for cloning of a gene inside (which is much harder than just cut and close it again) and this cloning worked fine, without any randomly plasmids afterwards.
I used this recombining plasmid as well just to transform E. coli DH5alpha and TOP10 to make Midipreps and no recombination takes place ... but as soon as I treat it in vitro, it's recombining.

My (easy) strategy:
- cut plasmid with XbaI
- heat-inactivate XbaI
- blunt with Klenow
- Phenol-Extraction
- Agarose-gel for band-extraction to eliminate any uncut plasmid
- EtOH-Precipitation to concentrate
- Ligation over night at 15 C

Thanks for your ideas.

#2 HomeBrew

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Posted 13 July 2010 - 04:42 AM

I would use site directed mutagenesis to eliminate the XbaI site, using a kit like Stratagene's (now Agilent Technologies) QuikChange II Site-Directed Mutagenesis Kit.

#3 astartus

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Posted 13 July 2010 - 05:30 AM

I would use site directed mutagenesis to eliminate the XbaI site, using a kit like Stratagene's (now Agilent Technologies) QuikChange II Site-Directed Mutagenesis Kit.


yes, could work much better. But the problem is: If such a simple thing like cut-blunt-ligate is not working, then cloning a gene inside will never work. Another issue is, that the lab I'm currently working in is not the richest one - I think they won't buy a kit just do kill one restriction-site :)

#4 HomeBrew

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Posted 13 July 2010 - 08:11 AM

Well, you probably don't need to buy a kit -- just two primers and some high fidelity polymerase would likely do the trick. Do the PCR, add XbaI to the reaction mix after PCR and allow the remaining wild-type plasmid to be linearized, and transform E. coli. If you add up the cost of all the stuff you're now using and multiply by the number of times you've tried the experiment, you'll probably save money...

#5 astartus

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Posted 15 July 2010 - 12:35 AM

Well, you probably don't need to buy a kit -- just two primers and some high fidelity polymerase would likely do the trick. Do the PCR, add XbaI to the reaction mix after PCR and allow the remaining wild-type plasmid to be linearized, and transform E. coli. If you add up the cost of all the stuff you're now using and multiply by the number of times you've tried the experiment, you'll probably save money...


Thanks Homebrew! That could be a solution for my problem ... but, the neighbour-lab is using the plasmid for cloning a gene inside and in their case, it's also always recombined after trafo of colis.

so, I will do cloning as well and then, I'm going to face the same problem. With or without XbaI-restriction site.

Has anybody any idea, what could be the problem?

#6 HomeBrew

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Posted 15 July 2010 - 04:57 AM

Let me see if I can get this straight...

Is it:

1.) The plasmid you're using is stable in E. coli when there is no insert (vector only), but as soon as you clone in an insert, it becomes unstable and undergoes recombination.

or

2.) The plasmid you're using is stable in E. coli with or without an insert, but it becomes unstable and undergoes recombination if you try to change the ends using Klenow and re-ligation.


If it's 1, then either the site into which you're cloning resides in a gene or regulatory element crucial to plasmid stability, or your insert encodes something that interferes with plasmid stability, or your insert is toxic to E. coli, resulting in recovery of only recombinants that have lost the toxicity.

If it's 2, then your treatment with Klenow is too aggressive, and you're chewing away way too much, resulting in a population of plasmids with variously truncated inserts, which look like recombinants.

#7 astartus

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Posted 21 July 2010 - 02:37 AM

Let me see if I can get this straight...

Is it:

1.) The plasmid you're using is stable in E. coli when there is no insert (vector only), but as soon as you clone in an insert, it becomes unstable and undergoes recombination.

or

2.) The plasmid you're using is stable in E. coli with or without an insert, but it becomes unstable and undergoes recombination if you try to change the ends using Klenow and re-ligation.


If it's 1, then either the site into which you're cloning resides in a gene or regulatory element crucial to plasmid stability, or your insert encodes something that interferes with plasmid stability, or your insert is toxic to E. coli, resulting in recovery of only recombinants that have lost the toxicity.

If it's 2, then your treatment with Klenow is too aggressive, and you're chewing away way too much, resulting in a population of plasmids with variously truncated inserts, which look like recombinants.


Both your possibilities are right ... the lab next to mine tries to clone something inside and they are facing the same problem of always randomly plasmids.

I try just to eliminate one restriction site with cutting - blunting - ligating and I have the same problem. Klenow ... mmmh, I thought about that as well. But: After Klenow I do Phenol-Chloroform and isolate the band from a gel ... and on the gel there is one band - no smear. If Klenow would be eating too much, wouldn't it be a smear?

thx




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