I tried Gibson Assembly to do cloning recently but all failed.
I used Snapgene to get primers for the amplication of vector and fragment. PCR worked well. Vector is 2.6kb and Fragment is 4.3kb. Notice the fragment (insert) is bigger than the vector.
I used 2X Gibson mix, always 50ng of vector, and tried Vector : Fragment = 3:1 and 1:3, since fragment is bigger than vector. The incubation time I tried 50C 15min, 25min, 60 min. The transformation I tried electrocompentent cells, stabl3 cells, and One shot cells.
I smeared all bacterial onto the selective plate for colony growth.
I could find a few colonies on the plate, but the sequencing results showed they are all original vectors.
Then I further used Dpn I to digest the Gibson reaction products before transformation, and the subsequent sequencing results of the colonies showed that they are the self-circled constructs of vector PCR product.
Anyway, the Gibson assembly did not work. Why? because the fragment is too larger? Do I need to try vector : Fragment=1:1? Do I need to extend the incubation time for Assembly, like 2-3 hours? The overlap of primers is 40 bp.
Thank you so much for your suggestion. I am looking forward to your opinion.
Qinhong Wang
UNC-Chapel Hill
Hard to provide an exact answer without knowing the details. But my checklist is usually as follows.
1. Recheck your antibiotic plates – easy mistake that we all make is plating onto the wrong antibiotic.
2. Is your marker AmpR or KanR? With KanR you should recover your bacteria in LB (or whichever non selective media you like) after you electroporate – I usually do this at 30˚C for 1 hour.
3. Check that your overlapping sequences are correct. I always do an in silico Gibson assembly in SnapGene. That way you can rule out a design issue.
4. Ensure your Gibson parts are good quality. Gel purify your insert. Gel purify your plasmid backbone. If the plasmid backbone was digested always incubate with CIP (Alkaline Phosphatase Calf Intestinal), you can usually do the digestion + CIP together (ie cut smart buffers).
5. Try diluting your Gibson mix 1:10 in water (autoclaved, nuclease free) and transform the diluted mix. Similar to this, you can dialyze your Gibson mix (https://www.sigmaaldrich.com/US/en/product/mm/vswp01300). In any case if you are electroporating your bacteria removing salts from your DNA is key.
6. Grow your e. coli at 30˚C and not 37˚C. Sometimes the insert is toxic at higher temps.
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I do not think the issue is that the insert is larger than the vector (esp. for sizes
Thank you so much for your suggestions. I think they are all useful. I will try to do based on your instructions. Will let you know.
Qinhong
How long do I need to incubate for Gibson reaction at 50C? 30 mins is enough? or 60 mins?
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