Table 1.
The efficiency of fragments assembly
| Fragments | Size (kb) | Plasmid sizesa (positive/ tested) | Verification of positive clonesb (correct/checked) |
|---|---|---|---|
| PCR amplicons | |||
| Vector + A | 6.5 | 30/30 (100%) | DNA sequencing (S) (10/10) |
| Vector + A + B | 10.5 | 26/30 (87%) | S (10/10) |
| Vector + A + B + C | 14.6 | 17/30 (57%) | S (10/10) |
| Restriction fragments | |||
| pSCactI-II | 18 | 22/30 (73%) | S (10/10) |
| pSCactIII-IV | 15.5 | 15/30 (50%) | S (10/10) |
| pHIW | 36.3 | 2/30 (6.7%) | BamHI restriction (2/2) |
aTransformants were first checked by the plasmid sizes and the efficiency was shown in the bracket.
bExcept plasmid pHIW, 10 positive clones of each construction were chosen for DNA sequencing verification, among which five clones were sequenced with primer Mseq-1, whereas the other five with Mseq-2 (Supplementary Table S1). Based on the sequencing results, the DNA sequences of the ligation sites were correct in all tested clones. Plasmid pHIW was verified by the BamHI restriction (Figure 3D). Two obtained clones with the desired sizes were proven correct based on the BamHI restriction analysis.