I am trying to perform a hifi assembly(NEB HIFI master mix) of a backbone (5.7kb) and inserts ( 0.9 - 1.5 kb) in a single fragment reaction assembly ( a different construct corresponding to each insert and not all of them together). My PCR amplification is fine and i get pretty good yields and good 260/280 and 230/260 ratios after gel extraction using the Zymo gel extraction kit. My backbone yields are low but around 30 ng/ul and decent 260/280 but 230/260 was lower around 1.3 ( so , the ratio was really bad after gel extraction (0.06) and i had to use the clean and concentrator kit from zymo to remove contaminants and the resultant ratio was 1.3). my vector is in a pET28b backbone and i performed Single digestion using BamH1-HF and then gel extracted the band. After this, I added my insert and the backbone ( backbone : 100ng, insert is 30.91 ng, calculated using NEB calculator as pmole. Insert : vector ratio is 1:2.) I do not get any colonies on my test plate. I was worried about self-ligation but that does not happen as self-ligation of the backbone would have lead to background colonies having no insert but that wasn't the case. My primers have roughly 20 nt long overhangs complementary to the backbone on either side of the gene of interest. I ran the hifi assembly for 15 minutes at 50 as suggested in the protocol. I have transformed using NEB cells and followed their protocol as well as used home-made DH5a comp cells and transformed a larger volume of gibson mixture. Is the backbone and/or the pcr amplicons lacking in the overhang? using IDT i analyzed any chance of any of the primers forming hairpin or other secondary structures and found that my reverse primer overhang ( complementary to the backbone) does form hairpin with a Tm of 49.3 which is pretty close to 50C for the gibson. Does this affect the efficiency of the cloning process. I have tried this two times , once each with gibson and hifi reaction mixture and both times they were unsuccessful. With the gibson, i had used a different backbone but same inserts. In that case, i had performed double digestion of the backbone using ndeI and xhoI. I had gel extracted then as well and done the gibson for 60 min at 50 without any success. I think i am missing something that went wrong in both cases, but dont know what. Any help would be appreciated.Thanks!
I think you have multiple problems there:
1- Even with failed assemblies you should get a background of miss-assembled colonies. The fact that you are not even getting any colonies (even self ligated clones which you should get many of them) indicates your transformation is at fault here.
2- You are trying to gibson assemble into a multiple cloning site and this is known to be difficult (it is still doable).
try the following:
1-with the reaction you currently have dilute 1:10 then add 2ul to transform neb-10-beta (the gibson mix is very salty and adding more it will decrease how many transformants you get and not increase it). If you would like to add a larger amount of your assembly you have to do ethanol precipitation first.
2-Repeat the reaction with 60 minutes reaction time. You can also setup a reaction at 55C to help with the secondary structure.
3-try extending the homology to 30bp.
4-digest the plasmid with XhoI and BamHI and ligate a 100bp adapter that has a simple sequence (derived from the 3' end of your insert) into the plasmid and redesign your primers to have homology to the adapter rather than the multiple cloning sites. Digest with BamHI and then do the assembly as above.
5- if all fails you could try to use standard ligation instead.
Good responses from Hanna. You can't even know that there's a problem with the assembly, before you've fixed your transformation to the point where you get dozens of colonies. It's entirely likely that once you do that, some of them will be correct.
Follow Hanna's other suggestions only when 8 out of 8 colonies tested are undesirable. The only thing I'll add is that you could use a higher insert:vector ratio. 1:5 is fine for inserts this small.
Hi everyone, this failure was a result of using a non toxic gel staining dye as a substitute for ETBr. Reverting back to ETBr gave successful Gibson's, not just for me but for the whole lab in general.
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