2. Easy gene assembly using multiple gBlocks Gene Fragments
Two or more gBlocks Gene Fragments can be easily assembled using the Gibson Isothermal® Assembly to generate larger DNA sequences . The fidelity of gBlocks Gene Fragments substantially reduces the number of clones that need to be sequenced to identify the intended construct when compared to other methods of gene assembly using DNA oligonucleotides.
The EGFP and kanamycin genes were assembled from either 2 or 3 gBlocks Gene Fragments, respectively. Each gene was divided into approximately equal size gBlocks Gene Fragments with 30 bp sequence overlaps at the ends of adjacent fragments as is required for the Gibson Assembly method . The fragments were then assembled into a pUC57 vector. The results show that in most cases, when assembling 2–3 gBlocks Gene Fragments, sequencing as few as eight clones is sufficient to find a wild-type sequence; more challenging sequences and complex designs may require sequencing of additional clones. For more information selecting clones, see Tips from the Bench below.
Table 1. Multiple gBlocks Gene Fragments® can be assembled using the Gibson Assembly® method to easily generate larger, high-fidelity DNA sequences.
|Wild-type Clones ||Gene length (nt) ||# gBlocks Gene Fragments || Correct clones* |
|EGFP || 720 || 2 ||3/8 |
|Kanamycin ||816 ||3 ||2/8 |
* Verified by double-stranded DNA sequencing
Experimental Details: Kanamycin and EGFP genes were assembled from 2 or 3 gBlocks Gene Fragments, respectively, and cloned into a pUC57 vector, linearized with EcoRV, using the Gibson Assembly™ method . Isothermal assembled plasmids were transformed into XL1Blue cells and resulting clones were verified for the assembled genes, using traditional Sanger sequencing.
Tips from the Bench
gBlocks Gene Fragments are produced using our highest fidelity synthesis methods. However, the chance of a single error affecting a final assembled molecule increases with the number of fragments assembled. We recommend sequencing at least 2 x (number gBlocks fragments assembled) clones to give you the highest probability of successfully identifying your desired target. For example, if you assemble 4 gBlocks Gene Fragments we recommend sequencing 8 clones to have the best chance (95%) of obtaining your desired construct.
3. gBlocks Gene Fragments are compatible with traditional cloning methods and vectors
Because gBlocks Gene Fragments are compatible with all cloning methods requiring double-stranded DNA as a starting material, they have exceptional potential for designing and assembling your desired construct sequence into any favorite cloning system.
Table 2. gBlocks Gene Fragments are compatible for cloning in a variety of standard vectors, using common cloning methods (traditional blunt-end cloning and Gibson Assembly® methods are shown in Sections 1 and 2 above, respectively).
| ||Correct clones || Clones sequenced ||% wild-type clones |
|Restriction cloning || 27 ||32 || 84% |
|TOPO cloning || 3 ||4 ||75% |
Experimental Details: Single gBlocks Gene Fragments ranging between 223 and 296 bp were cloned into selected plasmids, including: pUC57, pBluescriptII, pET27, psiCHECK-2, Zero Blunt TOPO, pIDTSMART, pGEM T Easy. Resulting clones were sequence verified by double-stranded sequencing.
- Gibson D, Young L, et al. (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods, 6(5):343–345.