Dear All
How do I decide upon the proportions for Gibson Assembly. I have a linearized (digested) vector 2958 bps (27 ng/ul) in which I want to clone two DNA fragments. One fragment is small 410 bps (80.1 ng/ul) and another is big 2750 bps (66.2 ng/ul). I have a 15 ul 1.33X master mix for assembly and now would need 5ul of the vector+small fragment+big fragment. How should I decide upon the proportions.
I would appreciate your help.
Thank you
Ikram
Hi Ikram,
equimolar amounts for all fragments are recommended. For example if you use 100 ng of the vector (3.7 µl), this would be 14 ng (0.2 µl) for the small and 93 ng (1.4 µl) for the big fragment. But this mixture would exceed the maximum volume of 5 µl, so just scale down the amounts to fit to 5 µl. If the assembly won't work using these amounts of DNA, I recommend to scale up the amount of DNA input for the small fragment (try 2 times and 5 times higher amount).
Good luck
Gertrud
Hi Gertrud
Thank you very much. I have successfully cloned my fragments. Just confirmed with colony PCR. I appreciate your help.
Thank you
Ikram
Hi Ikram,
I have a similiar problem like you do. I would be glad if you could tell me about the number of base pairs for overhangs for each fragment. I was consulting NEB builder help, however it suggest me to add only 15 base pairs as overhangs. I wonder whether it would work. What do you think.
Regards,
Bikram
Dear Bikram
I have since successfully cloned the fragments. The overhangs can be from 15-25 to 70-80 bp long. I prefer overhangs of 20-40bps. Also, I would recommend you to use snapgene software, the trail version is free for a month and it allows you to build the constructs very nicely (simulated). It will design the primers by itself. You have to provide the vector sequences and the insert sequences and thats all.
I hope this helps.
Thank you
Ikram
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