I'm assembling 3 PCR fragments with my vector pBAD and transforming them into dh5a. The second PCR fragment has 4 variations to it and the template for this PCR fragment comes from a plasmid. I've purified my second and vector PCR fragment using a gel extraction method. My assembly reaction consists of my vector normalised to 0.012 pmol and my PCR fragments at a total of 0.024 pmol, each. I then used 5ul of the NEB HiFI assembly master mix and dnase free water to a total of 10ul reaction, incubated at 50C for 60 mins and used 2ul with 50ul cells of dh5a for a heat transformation and streaked the transformants on amp plates (100ug/ul). However, I get no colonies growing the next day.
I've repeated this assembly twice - just to be sure I'm not making any silly mistakes, but still get no successful colonies the next day. I'm stumped as to figuring what's going on. Any ideas?
What's the length of overlaps between your fragments, and what's the melting temp of those overlaps? Sometimes overlaps that are the "minimum length" recommended don't work, especially if they have a tm that's much lower than 50°C.
If it's not that...
The kit should include a positive control reaction, have you tried that? I've had NEBuilder master mix go bad after 6 months before. If that doesn't transform well then either the kit is going bad or your competent cells are.
I'd also try using a column clean-up kit on your reaction after it's done, elute in a low volume (or concentrate it down) then put the entire thing into your comp cells. Sometimes their 2 µL straight from the reaction doesn't work very well, especially if the assembly was inefficient.
The other thing I would try is to incubate your cells for longer before recovery, maybe 90-120 minutes instead of the usual 60. I think E. coli has a bit of trouble replicating Gibson Assembly products initially sometimes, it seems especially rough when my vector backbone is a digested miniprepped plasmid and the rest is all PCR products. (When all components are PCR products this happens less, no idea why.) Also after you do a couple plates with 100 µL streaked out on each I'd leave the recovery overnight at room temperature, then plate the whole recovery if you didn't get any colonies. I've had a couple cases where I got no colonies from the 90 minute recovery but got a lot from the overnight recovery.
Hello I think the percentage that the assembly DNA represent of your total reaction is the problem. Usually it should not be more the 20% and since you are using a low volume reaction you probably exceeded that. Redo the reaction with increased volume (20ul) as indicated in the kit and do not add more than 4ul of the DNA to be assembled. I also usually dilute my reaction 1 to 5 before transforming 2ul in (in case the undiluted 2ul did not yield colonies). Finally, DH5 is not optimal for this kind of cloning especially if you creating them in house. I would recommend you change to NEB-10-beta high efficiency cells if you can.
Hey Liyana,
first of all: You are not doing any silly mistakes, science can be a tough and frustrating job.
I'm not sure if I understood everything correctly. In general, a gel-extraction is the best way to purify your DNA-fragments for Gibson, to avoid any contaminations (especially from "wrong" DNA).
Further, the protocol recommends a 3:1 molar ratio insert:vector. DNA concentrations can be measured using a Nanodrop or you can load in onto an agarose gel and compare it with a quantitive marker. Both methods show some inaccuracies. So, a good option is, if your gibson fails, to try different molar ratios (1:2, 1:5, 1:10; ...).
After a succesfull assembly I would use the whole Gibson - or at least more than 2 ul for transformation. If you get colonies you will throw away your assemly anyways.
Next, the transformation efficieny can be a problem, too. Like Hanna said: commercial cells can be a solution. I would include a positve control (something like 50 ng of pUC19 plasmid).
One last important point and maybe the most important. Have you checked you primers and overhangs?
I hope you got some transformants containing the correct assembled (and mutation-free) plasmids!
Best,
Jonas.
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