Transformation Results Flashcards
what is the purpose of a technical control?
make sure technique is correct/getting expected results
what is the purpose of an experimental control?
represents the “normal” condition so that experimental conditions can be compared to this, not always present (ex. comparing different plasmids wouldn’t need experimental control, but if it is certain conc, exp. control would be 0 conc)
what are the technical controls used in our experiment?
- H2O: ligation technical control
- plasmid only: transformation technical control
- TE buffer: transformation technical control
- TE diluted buffer: transformation technical control
what is seen on the sample of interest: insert + plasmid plate? why?
type of plate used?
- white colonies (ligation successful)
- blue colonies (ligation unsuccessful)
- all colonies that appear had successful transformation of plasmid bc amp resistant
LB+amp agar
what is seen on the H2O plate? why? purpose of plate?
plate?
- all blue colonies
- expect 0 white bc should be no ligation bc insert was not added
- shows only way to get white colonies is with insert presence
LB+amp agar
what is seen on the plasmid only plate? why? purpose of plate?
plate?
- UNCUT plasmid (no insert/no digest/no ligation)
- all blue colonies (bc no insert)
- ensures that transformation occurs bc it can survive with amp presence
LB+amp agar
what is seen on the TE buffer plate? why? purpose of plate?
plate?
- no plasmid was added to bacteria = expect 0 colonies bc bacteria can’t grow without ampR
- makes sure we don’t get growth when unexpected
LB+amp agar
what is seen on the TE buffer plate? why? purpose of plate?
plate type?
- no plasmid = not antibiotic resistance but makes sure that the bacteria are alive after transformation protocol
- we need dilution bc then we get countable bacteria bc w/out diution we’d get lawn/confluent growth
LB agar
If any of your technical controls do now show what is expected…
you cannot trust your samples
What if plasmid does not ligate to insert or to self?
- Unligated plasmid = linear
- Exonuclease degrades linear DNA of unligated plasmid from end so no longer in plasmid
transformation efficiency calculation?
total white + blue colonies on insert plate/(total colonies from TE diluted plate x dilution factor)
how many transformed/how many bacteria there were before transformation
ligation efficiency calculation?
white from insert plate/all colonies on insert plate
how many bacteria ligated out of total present
