From 2011.igem.org

Gibson assembly efficiency assay

For each of the Gibson reactions, we added 20ng of gel-extracted PCR products for the inserts and backbones. For the pSB assay we used the primers for the inserts and () primers for the backbone.

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.
4. . * INS + BCK tubes (x 2)
   • • add 1 uL of 10X-diluted pGA Gibson product (1A3 BCK/INS pLacGFP)
5. . * INSctrl tube

    * add 100 pg of pLacGFP gel extract

6. * BCKctrl

    * add 100 pg of 1A3 gel extract

Repeat the process for the comparison pSB vector as follows:

pSB vector Assembly

1. • 1 uL of pSB Gibson product (T19-1A3/pLacGFP)
   • 1 ng INS (pLacGFP- gel extract)
   • 1 ng BCK (T19-1A3- gel extract)
2. Once all the samples are ready, begin the transformation.
   • Rescue each sample in 500 mLs of LB
   • Incubate all samples @ 37oC for ~45 min.
3. 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)