exchange biologists,

Currently having some trouble getting a Gibson assembly to work and as it's my first time working with this method I thought I'd ask for some advice.

See the bottom of this post for my plasmids/primers

What I'm doing right now is cutting the both the plasmid containing the insert and the one containing the backbone with two restriction enzymes each, and using primers designed through Benchling's Gibson assembly primer design function to generate the ends needed for Gibson assembly. For my PCR steps I'm using a combination of Q5-polymerase and Dreamtaq as recommended to me by my supervising postdoc. I've worked in 2bp spacers into my primers to ensure proper translation.

Sadly my gels don't show bands that indicate success, any helpful suggestions would be appreciated. I'm currently considering additional sets of primers and PCR to amplify my insert and backbone prior to PCR using the gibson primers, as well as trying to find out if I could be using more suitable polymerases.

FWD gacgagctgtacaaggaattGCATCAGCCCGTGGGAACTCATGA
REV ctcaccatggtggcgaccggGGCTGGCTCCAGCTCTCGGGAGAG

Assembly product (primers are also included in here): https://benchling.com/s/seq-TJW7zqQuje5SM4csCIQF

Backbone plasmid, to be cut with EcoRI+AgeI: https://benchling.com/s/seq-2myVyzw3zoXh9PTvhCxG

Insert plasmid, to be cut with EcoRV+NheI: https://benchling.com/s/seq-HO34u3Z3RPTV4UW2z7S4

For those new to benchling, note that the cuts can be visualised through the scissor button on the right side of the screen.

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