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Trouble with subcloning


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#1 poussin

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Posted 10 December 2009 - 06:58 AM

Hi everybody,

I'm trying to perform a subcloning into a vector containing Tol2 sequences (to allow insertion on the genome). I've already successfully cloned the same insert into a classic vector allowing expression into eukariotic cells with no special troubles... So I guess that my insert is not toxic for bacteria! But since I've tried to do it in this Tol2 vector, all things went wrong. We first suspected that it was something with this vector into classic DH5alpha, that's why we tried with Sure2 supercompetent cells. Unfortunately, the results are not better... I can obtain a few clones. However when I perform digestion, in the best cases, I can see the right band for my insert but the vector size is half the one expected. In other cases, without performing any digestion on the Miniprep, I see 2bands with lower size than the ones of the insert and the vector as if I had performed digestion... I don't understand anything!!! Moreover, transformation of the original Tol2 vector into DH5alpha or Sure2 bacteria is OK and I performed at the same time another subcloning in another vector which worked fine!
Is it possible that the association of this insert in this vector does something wrong in the bacteria?? Do you have any idea to solve this problem?

Many thanks in advance

A desperate subcloner...

#2 aimikins

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Posted 10 December 2009 - 12:43 PM

could you post of pic of the gels you're getting, with the standards marked, and show where it deviates from expected? uncut DNA usually gives you a couple smaller bands...plasmid DNA exists in several forms; might that be what you see with uncut?

and how do you prepare and purify your fragments?
"it is a miracle that curiosity survives formal education" -A.E.

#3 poussin

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Posted 10 December 2009 - 02:28 PM

I ve attached a ppt file with a picture of the gel with the insert and the vector and the gel with non digested DNA obtained from 2clones.
I know that plasmid DNA exits in several form that do not migrate at the right size when not digested, but here, it can not be the explanation... I've already performed different subcloning, but I've never seen something as strange as that!!!
I prepared my insert and my vector by digestion and gel purification.
If you have any other idea, it would be helpful!


could you post of pic of the gels you're getting, with the standards marked, and show where it deviates from expected? uncut DNA usually gives you a couple smaller bands...plasmid DNA exists in several forms; might that be what you see with uncut?

and how do you prepare and purify your fragments?

Attached Files



#4 aimikins

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Posted 10 December 2009 - 05:37 PM

hm. the undigested DNA seems to suggest that you got a smaller clone than your target. #1 and #2 are most certainly uncut plasmid, it's just possibly not what you were looking for.

what happens when you linearize your clone?

since both your test clones look the same, probably there's something wrong in an upstream step, right? so my best guess is that you do not have the fragments you think you have, no matter how the gel looked before purification.

the insert isn't something that can self-ligate and be viable, is it? usually the lower plasmid DNA (uncut) runs ~ 1/2 the size of the plasmid, give or take.

hmmm....this is a puzzle. could be DNA from outerspace, but probably not. are you absolutely certain of the purity of your stock of vector and insert?
"it is a miracle that curiosity survives formal education" -A.E.

#5 poussin

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Posted 11 December 2009 - 02:46 AM

I've tried to linearize my clones with an enzyme into the vector and another one into the insert, but it could not cut anything.
The insert doesn't contain the ampicillin resistance, so it should not be viable alone...
Concerning the purity of my vector and insert stock, I'm pretty sure that there isn't any contamination. Digestion to prepare insert and vector didn't give anything else than the expected bands. Moreover, I performed gel purification and checked my fragments on a gel before ligation. So if there is a contamination, I could not see it at any step of the subcloning...


hm. the undigested DNA seems to suggest that you got a smaller clone than your target. #1 and #2 are most certainly uncut plasmid, it's just possibly not what you were looking for.

what happens when you linearize your clone?

since both your test clones look the same, probably there's something wrong in an upstream step, right? so my best guess is that you do not have the fragments you think you have, no matter how the gel looked before purification.

the insert isn't something that can self-ligate and be viable, is it? usually the lower plasmid DNA (uncut) runs ~ 1/2 the size of the plasmid, give or take.

hmmm....this is a puzzle. could be DNA from outerspace, but probably not. are you absolutely certain of the purity of your stock of vector and insert?



#6 aimikins

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Posted 11 December 2009 - 11:39 AM

well, you certainly don't have what you were shooting for, though. I bet if you sequenced you'd find that it's something else entirely.

I'd start over from the beginning and try again.
"it is a miracle that curiosity survives formal education" -A.E.

#7 poussin

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Posted 14 December 2009 - 08:46 AM

I have already started over from the beginning, but it still doesn't work... I really need this construct, so I guess I don't have the choive: I'm going to do it again!
Thanks for the answer!

well, you certainly don't have what you were shooting for, though. I bet if you sequenced you'd find that it's something else entirely.

I'd start over from the beginning and try again.






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