TP53-3xFLAG plasmid backbone was gel purified using QIAGEN kit. MISO PCR products were run on 1% Agarose DNA gel and also gel purified.
15ul Gibson Master mix (acquired from Boeke Lab at NYU) and 5ul DNA (PCR amplicons and TP53-3xFLAG backbone in equimolar amounts) were combined in PCR tube and mixed by gentle tapping.
Tube was heated at 50C in preheated PCR block for 30 minutes.
2ul of assembly reaction was transformed into 100ul of XL-10 Gold competent cells.
After 3 failed attempts with NYU Gibson master mix , we succeeded with commercial Gibson mix from Quantabio:
20ul total reaction volume, ADD IN ORDER INDICATED!
- 11.23ul Nuclease Free H2O
- 2.0ul repliQa 10x Reaction Buffer
- 4.77ul DNA (0.37ul PCR product 1; 0.45ul PCR product 2; 1.03 PCR product 3; 2.92 PCR product 4)
- 2.0ul repliQa HiFi enzyme mix (cat# 95190-010)
Tubes were prepared on ice, then heated at 50C on PCR block for 1 hour, held at 4C until ready to use.
Transformed 5ul of reaction into 50ul XL10 Gold Ultra Competent cells.
Picked 30 colonies from plate (of hundreds of colonies) for mini preps
Of the 30 mini preps, restriction digest showed 5 plasmids with potential correct insert (looking for 2 corrected point mutations); these 5 were sent for sequencing. Of the 5 sent for sequencing, 2 had inconclusive sequencing results, 1 did not have the corrected bases, 1 had 1 base corrected and the other inconclusive, and 1 had both bases corrected. These last two were re-transformed into more XL10 Gold cells, plated and digested to re-confirm the Gibson Assembly worked. The clone (19Q) which initial sequencing showed one corrected base and one inonclusive, was still inconclusive on the DNA gel of the restriction digest. All 5 colonies picked from the other clone (2Q) appeared correct again on the DNA gel of the restriction digest. These 5 were sent again for sequencing, 4 were confirmed to have both bases corrected and 1 was inconclusive. These four were combined into one tube and this was used for transfection into HEK293 cells.
