IPTG
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IPTG added to the selective medium to artificially increase the expression
of the ampR gene (under control of lac operon)
increases transformation efficiency with this plasmid
explain why bacterial colonies containing re-circularized vector without insert should
not grow on the plate
Give this one a try later!
so there would be no interruption of the lethal gene eco471R, so the
bacteria would die
,inoculation
Give this one a try later!
cells from an isolated colony place in selective medium to start liquid
culture
must choose single isolated colony from the plate so the liquid culture
contains cells that all have the same plasmid
if choose multiple colonies, mixed DNA will not be useful for sequencing
2 methods of transformation
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heat shock transformation
electroporation
both methods require competent cells
Growing the cells in liquid culture
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optimal growth = E.coli need nutrients, oxygen, and warmth (37°C)
culture medium = Luria-bertani (LB) broth
shake bacteria at speed of 275rpm = needed to reach optimal bacterial
density
agarose gel
,Give this one a try later!
allows determination of the number fo PCR products, size of products,
success of PCR (intensity), presence of contamination and whether or not
there are primer dimers
if a different plasmid was chosen to do the sticky end cloning and the 3' ends of the
insert were left with sticky end A's, what would be the vector sequence that would
anneal to those A's
Give this one a try later!
you would have a T at the 5' end of the vector
explain why bacterial colonies that do NOT contain transformed plasmid would never
be expected
Give this one a try later!
because pJET has a control mechanism for transcription of ampR
we can grow colonies in ampicillin and only colonies with ampR will grow
incubating the cells
Give this one a try later!
, incubated in a nutrient medium for 1 hour to recover from transformation
and being to express the genes of the plasmid
explain why it is important to have a molar ratio for ligation that is 1:1 or more vector to
insert
Give this one a try later!
more efficient and optimal, ensures there aren't multiple vectors leading to
self-ligation does not occur and that there are not multiple inserts going
into the vector
degenerate primers
Give this one a try later!
multiple pairs based on consensus sequence
single stranded DNA fragments that go through PCR in order to amplify
DNA
fewer H-bonds if not 100% complimentary (increased annealing temp
breaks H-bonds)
increase chance of amplifying non-specific unwanted PCR products
the transformation buffer contains which of the following to assist the plasmid to pass
through the lipid cell membrane
Give this one a try later!
Give this one a try later!
IPTG added to the selective medium to artificially increase the expression
of the ampR gene (under control of lac operon)
increases transformation efficiency with this plasmid
explain why bacterial colonies containing re-circularized vector without insert should
not grow on the plate
Give this one a try later!
so there would be no interruption of the lethal gene eco471R, so the
bacteria would die
,inoculation
Give this one a try later!
cells from an isolated colony place in selective medium to start liquid
culture
must choose single isolated colony from the plate so the liquid culture
contains cells that all have the same plasmid
if choose multiple colonies, mixed DNA will not be useful for sequencing
2 methods of transformation
Give this one a try later!
heat shock transformation
electroporation
both methods require competent cells
Growing the cells in liquid culture
Give this one a try later!
optimal growth = E.coli need nutrients, oxygen, and warmth (37°C)
culture medium = Luria-bertani (LB) broth
shake bacteria at speed of 275rpm = needed to reach optimal bacterial
density
agarose gel
,Give this one a try later!
allows determination of the number fo PCR products, size of products,
success of PCR (intensity), presence of contamination and whether or not
there are primer dimers
if a different plasmid was chosen to do the sticky end cloning and the 3' ends of the
insert were left with sticky end A's, what would be the vector sequence that would
anneal to those A's
Give this one a try later!
you would have a T at the 5' end of the vector
explain why bacterial colonies that do NOT contain transformed plasmid would never
be expected
Give this one a try later!
because pJET has a control mechanism for transcription of ampR
we can grow colonies in ampicillin and only colonies with ampR will grow
incubating the cells
Give this one a try later!
, incubated in a nutrient medium for 1 hour to recover from transformation
and being to express the genes of the plasmid
explain why it is important to have a molar ratio for ligation that is 1:1 or more vector to
insert
Give this one a try later!
more efficient and optimal, ensures there aren't multiple vectors leading to
self-ligation does not occur and that there are not multiple inserts going
into the vector
degenerate primers
Give this one a try later!
multiple pairs based on consensus sequence
single stranded DNA fragments that go through PCR in order to amplify
DNA
fewer H-bonds if not 100% complimentary (increased annealing temp
breaks H-bonds)
increase chance of amplifying non-specific unwanted PCR products
the transformation buffer contains which of the following to assist the plasmid to pass
through the lipid cell membrane
Give this one a try later!
