Table 4.
Evaluation of a simple protocol without purification of PCR products in PCR cloning.
| Vector (Restriction enzymes for linearization) | Purification | White colonies (%)*a | Number of colonies*b | Cloning efficiencies*c |
|---|---|---|---|---|
| pCRZeroT (XcmI/XmaI) | − | 100.0 ± 0.0 | 8.0 ± 5.6 | 15/24 |
| pCRZeroT (XcmI/XmaI) | + | 100.0 ± 0.0 | 31.7 ± 7.4 | 17/24 |
| pCRZeroT (SmaI) | − | − | 483.7 ± 43.6 | 22/24 |
| pCRZeroT (SmaI) | + | − | 673.7 ± 55.5 | 23/24 |
| pGEM-T Easy | − | 10.8 ± 2.4 | 30.0 ± 10.5 | 17/24 |
| pGEM-T Easy | + | 17.7 ± 1.8 | 112.7 ± 12.1 | 21/24 |
The G6PDH1 (1.6 kbp) gene was amplified by KAPATaq EXtra DNA polymerase (for pCRZeroT (XcmI/XmaI)) or Tks Gflex DNA polymerase (for pCRZeroT (SmaI)) in a 50-μL reaction. pCRZeroT was linearized by digestion with the indicated restriction enzymes, and purified by a Gel/PCR Extraction Kit. The linearized pCRZeroT (50 ng) and each of the PCR products (±purification, 1/20 volume) were then ligated. Two and a half microliters of the 50-μL PCR reaction (Purification (−)) or 1 μL of 20 μL purified PCR-products (Purification (+)) was used for ligation. Three tenths of the ligation reaction volume was used to transform 30 μL of ECOS Competent E. coli DH5α chemically competent cells. Commercially available T-vector pGEM-T Easy was used as a control.
*aThe fraction of white colonies as a percentage of total colonies was calculated and expressed as mean percentage ± standard deviation of three independent experiments.
*bThe number of colonies is the mean ± standard deviation of three independent experiments. When pCRZeroT (XcmI/XmaI) and pGEM-T Easy were used as T-vectors, the number of colonies was represented as the number of white colonies.
*cThe cloning efficiencies are represented as “the number of clones with the confirmed correct length of insert DNA by colony PCR/number of white colonies subjected to colony PCR”.