Gibson assembly efficiency assay


The PCR reactions were conducted at 20 μL volumes with 2x Phusion Flash polymerase master mix, 1 ng plasmid template, and 0.5 μM of each primer.

For the pGA assay, the insert came from a pGA1C3 vector and the insert came from a pGA1A3 vector expressing GFP. We used primers pGAprefix_fwd and pGAsuffix_rev to amplify the insert, and pGAsuffix_fwd and pGAprefix_rev to amplify the backbone.

For the pSB assay, the insert came from a pSB3K3 vector and the insert came from a pSB1A3 vector expressing low GFP levels that are not strong enough to see visually. We used pre-existing Biobrick primers BioBrick_f and BioBrick_r to amplify the insert and Suffix_f and Prefix_r to amplify the backbone.

Gibson reactions

After performing PCR as outlined above, each fragment was run on a 1% agarose gel and gel-extracted using a Qiagen QIAquick gel extraction kit. 20 ng of gel-extracted insert and 20 ng of gel-extracted backbone were added to a 20 μL Gibson reaction which was set up on ice and incubated at 50°C for one hour.

pGA transformations

pGA vector Assembly

Note: Prepare these mixtures on ice

  1. Obtain a 40 uL aliquot of BL21 cells.
  2. Add 120 uL of ice water to the aliquot.
  3. The total volume is now 160 uL; split evenly among four labeled tubes: INS + BCK, INS + BCK, INSctrl, BCKctrl.
    • INS + BCK tubes (x 2)
      • add 1 uL of 10x-diluted pGA Gibson product (1A3 BCK/INS pLacGFP)
    • INSctrl tube
      • add 100 pg of pLacGFP gel extract
    • BCKctrl
      • add 100 pg of 1A3 gel extract

pSB transformations

Repeat the process for the comparison pSB vector as follows:

pSB vector Assembly
    • 1 uL of pSB Gibson product (T19-1A3/pLacGFP)
    • 1 ng INS (pLacGFP- gel extract)
    • 1 ng BCK (T19-1A3- gel extract)
  1. Once all the samples are ready, begin the transformation.
    • Rescue each sample in 500 mLs of LB
    • Incubate all samples @ 37oC for ~45 min.
  2. Plate 50 uL on each sample onto a LB + Chlor + Amp + IPTG plate.
    • 1 plate for each control in each vector set.
    • 3 plates for each Gibson product tube in each set. ( 6 plates for each vector set + 2 control plates)