Yeast lab Flashcards
what was the purpose of part 1 of the yeast experiment
isolation of yeast genomic DNA, restriction enzyme digest, and dephosphorylation of plasmid DNA
how was DNA isolated?
we extracted DNA from a yeast culture, and then used kits (with all the necessary buffers and columns) to isolate the DNA
what kit did we use to isolate DNA?
GeneJet Genomic DNA Purification kit
after lysing the cells, what did we treat them with?
RNase A
what does RNase A do?
removes the RNA
once the RNA was removed from our samples, what did we do?
used the purification column with its silica membrane
what is the purpose of the silica membrane in the column?
the DNA binds to the membrane
why do we wash the column with buffers?
to remove impurities
why don’t the buffers remove the DNA as well?
DNA is bound to the silica membrane
what is used to remove DNA from the column?
elution buffer
what plasmid was used as our cloning vector?
pUC19
what important things does pUC19 contain?
AmpR, ORI, and MCS
what is AmpR?
ampicillin resistance
what is ORI
origin of replication. Allows for replication in a bacterial host
what is MCS
a multiple cloning site that contains various unique restriction sites
how was pUC19 digested?
using the restriction enzyme EcoRI
what sequence does EcoRI recognize?
5’-GAATTC-3’
what does EcoRI do at the 5’-GAATTC-3’ sequence?
it makes a staggered cut between the G and the A to create complementary sticky ends
what is the final step of part 1?
dephosphorylation of the digested plasmid
how do we dephosphorylate the digested plasmid?
by using alkaline phosphatase
why do we dephosphorylate the digested plasmid?
to prevent recircularization of during the ligation reaction
what is the purpose of part 2 of the yeast experiment?
restriction enzyme digest of genomic DNA, ligation, preparation of competent E coli cells, transformation and plating
what does EcoRI do?
digests the isolated yeast genome into smaller restriction fragments
how do we digest the isolated yeast DNA into smaller restriction fragments?
by using EcoRI, the restriction enzyme