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Failed Gibson transformation

Gibson transformation

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#1 aadamson81

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Posted 28 October 2015 - 02:26 AM

Hi, I've been trying to clone three PCR fragments into a vector using Gibson cloning.

All three fragments amplified well with the chimeric primers, and used in the reaction onto a pUC19 background.

However I failed to get any transformants. I did PCR analysis from vector:fragment 2 and fragment2:vector and obtained strong bands at the predicted size for both fragments indicating the presence of the desired product. After speccing the sample the DNA concentration was quite high (~3000ng/ul) so I tried first diluting before transformation into both DH5a chemically competent cells, and DH10B electrocompetent cells and plating on Amp plates, but again both these transformations failed. Next I used plasmid safe exonuclease to digest remaining linear DNA, and then cleaned the reaction through a column, eluting in 30ul water. Speccing of this sample indicated I had a much reduced concentration (8ng/ul). I repeated the PCR test on this cleaned template and again obtained both bands at correct size, but still the reaction failed to transform.

It seems to me the product has been generated (as only circular DNA would survive the plasmidsafe digestion, and the PCR reactions were successful) but the lack of transformation is very puzzling. Do you have any advice?

Thanks

Antony



#2 Cutter

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Posted 14 November 2015 - 07:23 AM

Assuming that you can confidently rule out anything post-transformation (do you have a positive control?), my best guess is that there is not enough of the desired product. There is enough for a positive signal from PCR, but maybe not enough for transformation. The efficiency of Gibson assembly decreases as the number of fragment increases, and as the size of the fragments increases. How large are the fragments? Could the overlaps be forming secondary structures, reducing the efficiency of assembly? One option is to amplify the linear form of the desired product (using back-to-back primers), then phosphorylate/ligate and transform. That ligation reaction will surely occur at much higher frequency than the Gibson assembly reaction. 



#3 phage434

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Posted 14 November 2015 - 08:17 AM

This, in my opinion, is the major issue with Gibson assembly. When it works, great, when it fails, you have no clue as to why, and no way to determine what the problem is. I would make sure I had competent cells that work. This is the most common problem with cloning of all kinds. You should test your cells and measure competence at > 10**7 cfu/ug of purified plasmid DNA. Ideally it would be more like 10**8 cfu/ug.



#4 labtastic

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Posted 16 November 2015 - 07:43 AM

When it works, great, when it fails, you have no clue as to why, and no way to determine what the problem is.

 

^^ Molecular cloning in a nutshell.  wink.png

 

It is a frustrating "science", but cloning is simply a means to an end. Do what works, avoid what doesn't. 


Edited by labtastic, 16 November 2015 - 07:49 AM.


#5 phage434

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Posted 16 November 2015 - 12:20 PM

Well, with restriction based cloning you *do* have ways of figuring out why it fails. You can, for instance, test each ligation junction independently and know that each works (or doesn't).



#6 Cutter

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Posted 18 November 2015 - 09:14 AM

You can, for instance, test each ligation junction independently and know that each works (or doesn't).

 

aadamson81 did test the junctions by PCR. Am I missing something?



#7 phage434

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Posted 18 November 2015 - 03:59 PM

No indication that fragment1 and fragment2 were overlapped and amplified. But more importantly, Gibson is not PCR, so this tells you that a pcr assembly might work, but not that Gibson assembly would work. I can't pairwise test Gibson assembly reactions.



#8 Cutter

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Posted 18 November 2015 - 10:42 PM

PCR analysis from vector:fragment2 amplified a band of the expected size, which indicates that frag1 and frag2 were overlapped. Good point re: Gibson is not PCR. 

 

On a related note, I wonder whether fragments with sticky ends generated by restriction enzymes could be assembled by PCR e.g. during the initial denaturation step (thereby giving false positive signal when attempting to amplify fragments that are resistant to restriction digestion) . What do you think?






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