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Revision as of 20:54, 28 September 2011

Gibson assembly efficiency assay

PCR

The PCR reactions were conducted 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 Biobrick primers BioBrick_f and [BioBrick_r]Suffix_f and [Prefix_r] to amplify the backbone.

Gibson reactions

Starting with each

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)