Day 3

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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.


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)
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