Bio1AL_Fa09_lab3_prelab -...

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Unformatted text preview: Transformation
and
Enzyme
Pre‐lab.

You
must
show
this
completed
pre‐lab
to
your
GSI
at
the
start
of
lab!

 (Pages
64­90
[focus
on
69­76],
91­110
[focus
on
91­97)

It
is
due
at
the
end
of
lab.

Thus
you
can
use
your
 flow
chart
during
lab.
 
 Name

 
 
 
 
 

GSI
&
Sect
#

 
 
 
 
 
Station
#
 
 
 To
answer
these
questions
read
all
of
the
lab
material
on
constructing
recombinant
DNA
(pages
64‐90)
even
 though
you
are
only
doing
pages
69‐76
for
lab
3.

You
also
need
to
read
about
enzymes
(pages
91‐110).

Write
 legibly.

You
will
NOT
be
admitted
to
lab
unless
you
turn
in
this
completed
pre‐lab
at
the
start!

 
 1)

Define
transformation,
competent
bacteria,
ampicillin
resistance
and
“lawn”
of
bacteria.
 
 
 
 
 
 2)

Fill
in
the
following
blanks.

Be
specific
for
the
experimental
conditions
for
Part
1
of
the
enzyme
lab.
 Substrate
(_____)
+
Enzyme
(_____)

reacts
for
____
minutes.

The
reaction
is
stopped
by
the
addition
of
____.

Upon
 heating
reducing
units
react
with
the
added
_______
to
produce
a
brown
soluble
product.

The
absorbance
of
this
 solution
can
be
determined
using
a
spectrophotometer.

A
1µM
maltose
solution
has
an
Optical
Density
of
0.4.

 Therefore
a
solution
with
an
O.
D.
of
0.6
would
represent
a
_____
µM
Maltose
solution.
 
 3)

What
results
would
you
predict
if
you
added
DNS
to
your
reaction
tubes
(after
5
minutes)
and
then
measured
 the
O.D.
of
the
sample
without
heating
(at
or
greater
than
95˚C)?

Explain!
 
 
 
 
 
 4)

Outline
the
general
procedure
to
measure
the
optical
density
of
a
sample.

Include
zeroing
the
transmittance
 (on/off
switch)
and
the
absorbance
(with
the
blank)
of
the
spectrophotometer.

A
diagram
of
the
 spectrophotometer
may
be
helpful.


 
 
 
 
 
 
 
 5)

You
are
given
a
tube
of
spit
that
has
already
been
centrifuged.

Outline
how
you
would
make
the
following
 dilutions:
1)
1/10,
2)
1/100,

3)

1/1,000,

4)
1/10,000
and
5)
1/100,000.

You
have
the
following
items:
test
 tubes,
a
dispensette
that
dispenses
4.5
ml
of
a
phosphate
buffer,
and
a
P
1,000
micropipetter.

Diagrams
might
be
 helpful.
 
 
 
 
 
 
 
 6)

Describe
how
to
use
a
micropipetter
–
say
this
out
loud
pretending
you
have
a
micropipetter
in
your
hand
 (you
are
on
the
honor
system).

Diagram
the
window
for
withdrawing
500
µl
of
solution
with
a
P1000
 micropipetter.
 
 
 
 
 Pre­lab
continued
on
the
other
side.
 A15
–
Fall
2009
 7)

The
following
three
solutions
are
used
in
separate
transformation
reactions
(3
separate
experiments).

 Predict
transformation
results
for
each
realizing
that
a
small
number
of
bacteria
get
transformed.


For
each
cell
 in
the
table
indicate
the
number
of
colonies
(lawn
or
isolated)
and
the
color
(white
or
blue).
 
 
 Type
of
petri
plate
 Solutions
 LB
only
 LB
+
AMP
+
X‐gal
 No
plasmid
DNA
 
 
 PBLU
plasmid
DNA
 
 
 PBLU
plasmid
DNA
with
insert
in
B
gal
gene
 
 
 
 8)
Prepare
a
flowchart
for
part
I
of
the
enzyme
experiment
(refer
to
the
flowchart
for
the
photosynthesis
lab
for
 an
example).

Your
flowchart
should
be
useful
to
you.

It
should
be
complete
enough
that
you
could
use
your
 flowchart
to
do
part
1
without
the
lab
manual.
 
 
 Flowchart
­
Part
I
 A16
–
Fall
2009
 ...
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