Dear colleagues,

I ran a colony PCR to see if colonies contain the insert after the transformaiton with a Gibson assembly. The products are ~1810 bp (ORF+primer-added additional 300 bp). When I ran it on 1% agarose gel, the bands for both products were a little bit heavier than should be (by ~200 bp).

Is it something wrong with the product, or is it the effect of the agarose gel and voltage itself?

-------------------------------------Experiment setup-------------------------------------

Plasmid prep: a purified, restriction-enzyme digested and column-purified vector

Insert prep: PCR on the open reading frame adjusted for Restriction enzyme sites, column purified.

Gibson assembly: NEB 2X mastermix, 50C incubation for 15 min with 8 cyclesx5 minutes at 40, 45, 50'C for fidelity.

Transformation: NEB DH5alpha E.coli standard transformation protocol with 5 uL of Gibson assembly

Colony Propagation: overnight at 37C under kanamycin-50 selection agar.

Colony PCR: 50-uL of Q5 2X mastermix (25 uL Q5 2X MM, 22.5 uL water, 1.25 uL of each primer at 10 uM for a final concentration of 250 nM for each F and R primers, a touch of each colony and resuspension in individual PCR rxn), 94'C cracking for 5 minutes, followed by 34 cycles of: [30 Sec 94'C denaturation, 55'C annealing for 1 minute, 72'C extension for 1 minute], final extension of 72'C for 2 min and hold on 4'C

Electrophoresis: 1% agarose TAE 1X buffer with SybrSAFE gel, run in TAE1X buffer at 130 V, 400mA for 90 minutes and visualised using BioRad transilluminator.