Hello!
I have done three successful Gibson assemblies, so to simplify: I put together parts named 1+2, 3+4 and 5+6. Every part is about 1000-2000bp. Those assemblies worked and I could see the three new 1-2, 3-4 and 6-5 assembly-parts on agarose gel after i PCR them.
But when I try to mix and do an assembly on all these assemblies together (1-2 + 3-4 + 5-6) to create one big part, it doesn't work. Not even the previous Gibson parts are visible on gel after I amplify the DNA with PCR.
All I can see are fragments that are too small to be any of my parts. I've tried multiple times and the same small fragments, one is at 800bp and is the strongest stripe (this stripe was seen on the 5-6 gel but was not nearly as strong as it where now) when the whole assembly should be at 11000bp.
What can I do to try to succeed with this gigantic Gibson assembly?
Could there be secondary structures that interfere with the PCR-primers?
Do you mean that you have 3 inserts: 1+2, 3+4 and 5+6? And you want to assemble all the 3 inserts into 1 vector? Maybe you can check these:
1) Are the concentration of insert sufficient after purification?
2) Normally, if you want to assemble more than 1 inserts, it is recommended to use Molar ratio of vector: insert:insert:insert 1:1:1:1, but you can increase it to 1:2:2:2 to increase chance of assembly.
3) May be you can try Hot-Fusion method: Article Hot Fusion: An Efficient Method to Clone Multiple DNA Fragme...
I tried on 1.1kb vector with insert 1-2kb. It works well for me every time.
Hi,
How are you designing the primers for the assembly?
I always use the http://nebuilder.neb.com/ on line tool to design primers.
I have been able to perform complex assemblies using this software to design primers. I was able to assemble a final l12 kb vector using 6 different parts ranging from 4 kb to 800 bp. Another thing is that you need to be careful with the proportions of the fragments in you assembly mix. For 4-6 fragment assembly it is absolutely essential (for high efficiency) that you have equimolar amounts of all fragments. For 2-3 fragment assembly, you can use 2:1 vector insert ratio.
The source of the assembly mix might also be a problem. I have heard from colleagues in the lab that in house prepared mixes are less efficient for complex assemblies (4-6 fragments, final assembly > 10 kb). If you failed with in house prepared mix, try the Hi-Fi assembly mix from NEB. It always worked for me.
All the best
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