Day 3
2.Outcome #1, no colonies on any plate: This would suggest that the transformation was unsuccessful, because even the positive control with the un-engineered M13K07 DNA failed to grow in the presence of kanamycin, meaning the DNA was not taken up by the cell.
Outcome #2, thousands of colonies on all plates: This could indicate contamination because the cells transformed with the negative control for ligation, backbone only and no ligase, should not have kanamycin resistance.
Outcome #3, approximately the same number of colonies on the backbone+ligase+kill cut as the backbone+insert+ligase+kill cut: This could also indicate that there wasn't complete digestion during the kill-cut and some backbone managed to back-ligate and be transformed into the cells of both samples.
3.
plasmid with insert | plasmid no insert | |
Enzyme(s) used | EcoRI, BmgBI | EcoRI, BmgBI |
Buffer used | NEB 3 | NEB 3 |
Temperature | 37 C | 37 C |
Predicted fragments | 8 bp, 8672 bp | 8680 bp |
Diagnostic digest 2 | ||
---|---|---|
Enzyme(s) used | BamHI | BamHI |
Buffer used | NEB 3 | NEB 3 |
Temperature | 37 C | 37 C |
Predicted fragments | 8680 bp plasmid and supercoiled | 8680 bp (linear) |