Dear all,
I did assembly of of five different DNA fragments having length (A= 975 bp, B=950 bp, C= 748 bp, D=1022 bp, Vector backbone= 1790 bp). The concentrations of these gel purified DNA fragments were A=57.1 ng/ul, B=117.5 ng/ul, C=31.6 ng/ul, D= 29.8 ng/ul, vector backbone= 151.1 ng/ul.
I used 0.25 pmol of each DNA fragment (i.e= A; 2.21 ul, B; 1.31 ul, C; 3.845 ul, D; 5.57 ul, and vector backbone; 1.92 ul respectively) for Gibson assembly. Total volume of fragments was 14.873 ul, so I added 2X 14.873 ul of GIBSON master mix. Therefore, final reaction volume was 29.7 ul. After mixing all the components together, it was incubated at 50'C in PCR. Lid temp was 51'C, and incubation was provided for 1 h at 50'C.
Following assembly of fragments, chemically competent cells of E.coli DH5 alfa were transformed, however, i did not found any colony. In vector backbone, ampicillin resistance cassette was present, So i added 100 ug/ml of ampcillin to LB agar media for screening of transformed colonies.
Primers I checked, they are with correct overhangs (20-30 bp). So no issues are with overhangs. Please give your opinions where I did mistake.
At the moment you have many areas in which the issue might lie and as such you need to narrow your options.
In the first instance it might helpful to repeat the reaction and run it on gel just to see how complete/incomplete the reaction has been. To ensure that you can see the smaller bands at this concentration run the samples on an E-gel/other thin gel system/bioanalyser......if you have access to one!!
Did you run the Gibson kit control?
As with any cloning technique purity of fragments is key so perhaps include additional wash steps with whichever clean-up kit you are using.
If the issue persists perhaps try ligating pairs of fragments to see if there's an issue with one fragment in particular.
Thanks Marcus,
I did Gibson control experiments. Kit is working properly.
Side by side I am trying to assemble the fragments A, B, C, D and vector backbone by overlapping PCR and Phusion polymerase. However, l got results for AB, CD assembly individually, but not for ABCD. So what u suggest, overhangs between B and C is not working properly? Should I change reverse primer of B and forward primer of C. I checked all these overhangs and junctions. They are OK.
One more thing, as u suggested to run the assembled product, I did it through agarose electrophoresis. I could not predict results. Because it was a huge smear. What u suggest, the assembly of 5 fragments is not possible with Gibson kit? I used Quiagen kit for gel elution from agarose gel.
If all the combinations work correctly (did you assess Vector+A, Vector+B separately as well?) except B+C then yes it would suggest some random secondary structure issues inhibiting the ligation at the B/C junction. Perhaps try varying amounts of DMSO which can help with secondary structure issues in PCR. Whether DMSO would be appropriate for this method I don't know, I haven't actually done this, I'm just trying to think of anything that might overcome your issue.
If there's something intrinsic to the B/C junction that's causing the problem then I would design two new primer sets (give yourself options in parallel), one for each side of the junction and just use 5' linkers to bridge the current junction.
With regard to the smear, is it at a high MW? If so, it could be concatemerisation and a two step protocol (A+B+C+D then ABCD + Vector) might be more appropriate.
Marcus, can you please explain what you mean by the "concatemerisation and a two step protocol" ? I'm having issues with my cloning using Gibson Assembly protocol as well. I'm trying to ligate 3 fragments (two obtained via PCR (i.e. A & B) and a digested vector (C)). Despite getting lots of colonies, only the smaller fragment seems to get inserted (I know this from sequencing). When running the Gibson assembly product on DNA gel, I can see a new band with an approximate size corresponding to fragments A+B, and I'm not sure how to troubleshoot this.
Marcus Hanley
Hi Angela,
How much nucleotides you have given for gibson overhang in fragments A & B?
As i realised, a directly purified clean PCR product using column (if single band obtained during amplification), and doing ligation in ratio 1:5 can largely help in getting lositive clones.
I would suggest you to to set a PCR, followed by direct clean up for insert, and clean up of vector backbone after Dpn1 digestion.
Secondarily, set a gibson rxn having all the inserts 5 times higher conc. than vector backbone, keep maximally for 1 h at 50 degree.
It should assemble properly. In my case it worked efficiently.
All the best.
I have 18 nt overlap between A & B. I did DpnI digestion after PCR reaction, then cleaned up the PCR product.
I'll try the 1:5:5 ratio of vector:A:B. Did you use molar or mass ratio?
Thank you :) @ Vikas Kumar Patel
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