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Plasmids recombining


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#1 starbright999

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Posted 03 January 2015 - 10:12 AM

I'm attempting to make several different constructs and they all seem to be recombining. The strategy I'm using is straightforward: I  digest my vector and insert, CIP treat the vector, gel extract both, ligate overnight, and then transform into DH5 alpha. My negative control transformation (ligation done with the vector only) is always negative, and I usually get plenty of colonies on the plates with cells transformed with the vector/insert ligation. However, when I miniprep the colonies the resulting plasmids are very small. I expect the resulting plasmids to be over 8 kb (depends on insert), but for these minipreps the most abundant band is at about 1 kb. When I miniprep the original vector I get bands of the expected size.

 

When I attempt to digest the minipreps, the only enzyme that cuts is one which should cut in the ampicillin resistance gene. When I sequence using vector specific primers, most of the primers don't prime and those that do give very short reads (100 bp or so).

 

I think that this is probably due to the plasmids recombining to delete not just my insert, but also most of the rest of the plasmid. Does anyone have any thoughts on ways to get around this? I have tried using different competent

cells (XL10-Gold and Stellar), incubating the plates at room temperature rather than 37, and incubating liquid cultures for minipreps at room temperature. No success. 

 

One thing that surprises me is that I'm having this problem with ALL of the constructs I'm making, even though they contain different combinations of vectors and inserts. I know that some sequences are unstable and E. coli doesn't like to make them, but it seems particularly unlucky that this is the case for all the constructs I'm working on.

 

I hope someone has some advice for me! 



#2 phage434

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Posted 03 January 2015 - 10:37 AM

Have you tried the control with just the insert DNA? Possibly your insert (depending on where it comes from) has a plasmid backbone that can transform. CIP treatment of vectors often causes problems. I would switch to shrimp alkaline phosphatase or antarctic phosphatase, and heat kill it. If you treat your vector digestion this way, there is no need to gel purify, assuming you can heat kill your enzyme (the insert cut out won't ligate back).



#3 Trof

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Posted 03 January 2015 - 10:48 AM

It's weird that happens to all of your cloning combination.

 

Some vectors are designed to carry a retroviral sequence to be later used as a source for a artificial virus particles.These recombine really very often and specific E. coli strains exists for cloning these, like Stbl2 and others from Invitrogen or SURE cells from Agillent. Other companies may have other strains for cloning unstable vectors.

 

Some inserts can have similar problem, containing repeats, so your vector could be fine, but the insert is not. In other cases, the insert is just killing the bacteria, co it must get rid of it. In these cases, the vector should not contain bacterial promoter. Also specific strains exist for cloning toxic inserts.

 

Also, bigger plasmids create more pressure on bacteria, so if you have a large insert and very high-copy plasmid, it may not work well together.

 

But unless all of your vectors are containing repeats, or all inserts, there seems to be more likely a problem with your procedure somehow. You say you gel extract it, is it possible that you actually fragment the plasmid, by UV exposure and the fragments reconnect since they are not CIPed, and of course the only one that stays in the cell is the one with AmpR and ori. If you can rule out all of your vectors contain repeats, try bypassing the gel extraction.. you may get more selfligation, but then you will see if you also have some correct inserts as well.

Other option is as many did mention on the forums, to actually PCR out the whole vector instead of cutting it, there would be the protocol for that somewhere.


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#4 starbright999

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Posted 03 January 2015 - 11:16 AM

Thank you both for the helpful suggestions!

 

The inserts are digested PCR products, so I don't think that's the problem. I am starting to think the problem is with my procedure though. I'll try switching to SAP and skipping the gel extraction and see if that helps. Otherwise, I'll give SURE cells a shot. And of course, continue praying to the cloning gods...






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