||2.27.07 Ligation and Transformation Data
|DNA Ligation Sample
||Expected Number of Transformants
||Observed Number of Transformants
| Control Plasmid||Many||1184
| BKB with cocktail but no ligase||0||0
| BKB with ligase and cocktail||0||6
| BKB with insert and ligase and cocktail #1||A few||0-1
| BKB with insert and ligase and cocktail #2||A few||5
2.27.06 Ligation and Transformation Data
We didn't determine the concentration of the prepped DNA from the ligation reactions, so we cannot calculate the transformation efficiency of these transformations. We do know, however, that 5 ng of vector was used to transform the control plasmid, so efficiency = (1184 colonies)/(5*10^-3 micrograms of DNA)= 5.92*10^6 colonies/microgram of DNA.
The control plasmid transformation serves as a positive control. It should work. If this transformation didn't work (along with some or all of the others), we might suspect an issue with the competency of the cells or with the transformation process. The appearance of colonies on this plate, however, demonstrates that this is not the case. The cells are indeed competent and our protocol worked.
The backbone with and without ligase mixed with the killcut cocktail serve as negative controls. They should not work. If a significant number of colonies were to show up on these plates, this would first mean that the restriction endonucleases weren't cutting. This could be because they had expired or because the sites no longer existed in the DNA. Assuming the latter for the ligated vector, this would mean that the vector sequence was somehow modified during the initial digest. For the linearized vector, the appearance of colonies would suggest that the DNA somehow became ligated and lost restriction sites, or the cell accepted cut vector.
The controls do not provide a definitive diagnosis of the problem should the experimental ligations fail, but they provide starting points for the inquiry into the failure.