Dear all,
I am currently trying to do a 3-way Gibson Assembly using 3 DNA fragments of size, 2.4 kb, 4 kb and 6.3 kb. The final ligated product will have a size of 12 kb. My protocol involves using equimolar of all these fragments (6.4 x 10^-5 nmol) with the addition of 4 uL of the commercial 2x Gibson Master Mix (NEB). No water is added into the reaction mixture. The reaction mixture was incubated at 50 degree Celsius for one hour. However, I haven't been successful in constructing the 12 kb product. Does anyone have any trick that I can go about to building this large DNA construct? Many thanks in advance.
Dear CheeHoo
Did you prepare purified PCR products? I have used the Gibsom assembly kit from NEB and the protocol says that "Column purification of PCR products may increase the efficiency of both high-fidelity DNA assembly when assembling longer than 5 kb fragments.
or How about assemble the fragments sequentially? like A-B -> AB-C -> ABC
c.f) I've never tried assembling more than 8 kb.
Dr. Daniel Gibson has used this method to assemble entire genomes, 12 Kbp may not be the issue here .
- Have you checked if the ends of the fragments for stable secondary structures at 50 °C ? 2° structures inhibit the subsequent elongation, annealing and ligation steps. it is not enough if the primers don't form stable 2° structures, check for 2° structures about 100 bp of termini.
- if there are no 2° structures, follow the suggestions by Jae-Seong Hwang.
- And if that fails too, or if there are 2° structures, i suggest you to try incubating at 50°C for 3-5 min, followed by incubation at 60°C, the exonuclease should do its job in 3-5 min, and the other two enzymes are from thermophilic organisms so they should retain their activity at higher incubation temperatures.
Dear Jae-Song,
Yes, I am using purified PCR products to do my Gibson ligation. I have also thought of doing the Gibson Assembly in sequential manner as you suggested. However, the ligated A-B product will have 3' overhangs and impossible to amplify since the T5 exonuclease had severed the 5' priming sites. Alternatively, I have tried doing an overlap extension PCR but hasnt been successful in my hands.
Dear Vamsi,
Yes, I have checked the secondary structures of the 60 bp overlapping regions that I am using. They dont seem to form a very strong secondary structure and should be alright for ligation. I will definitely try the modifications that you suggested for the assembly protocol. Thanks.
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