Day 4

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1.

Transformation Expected Results Actual Results Transformation Efficiency Explanations
Positive Control (pUC18) most colonies 300 colonies (300 colonies)/(5 ng plasmid DNA)=60,000 colonies/ μg DNA The positive control should have the most colonies because it is a well known plasmid that should not have anything encoded that will affect cell growth.
backbone only few if any 0 colonies 0 colonies/ μg DNA There should be little growth here because the backbone has been digested and purified, and no ligase was added to religate it. The bacteria usually do not read linear DNA.
backbone + ligase few if any 0 colonies 0 colonies/ μg DNA The growth of no colonies here suggests that our kill-cut digestions were successful in destroying all M13K07 backbone that may have religated with itself instead of the insert in the transformations below.
backbone + insert + ligase many colonies 0 colonies 0 colonies/ μg DNA The fact that we did not see any growth here indicates that perhaps our ligations were unsuccessful. This could be due to multiple things. For instance, our enzymes and buffers could have been bad. Also, because it was hard to see the pellet of DNA during purification, the backbone DNA may have gotten washed out or was not properly resuspended in water and remained lodged to the bottom of the eppendorf tube. Another possibility was that our insert was not designed properly and could not anneal or ligate with the backbone. The insert may also have coded for something lethal to the cell.
backbone + insert + ligase many colonies 0 colonies 0 colonies/ μg DNA see above
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