Download GFP Lab Manual.

Green Fluorescent Protein II:
Bacterial Transformation
Objective
To get some experience with recombinant DNA techniques and to genetically alter a bacterial
strain to produce a particular protein.
Background
Note that we have not covered this material in lecture yet, so, for today, you will need to do
some things without completely understanding them. Even if all the details are not clear, the
big picture is based on some pretty simple extensions of the genetics you already know. As the
course continues, what you have done today will be clearer.
Green Fluorescent Protein (GFP) is a protein produced by the jellyfish Aequoria victoria. The
protein fluoresces green when exposed to ultraviolet (UV) light. The goal of today’s lab is to
change the genes of a bacterium so that it now makes GFP and will fluoresce in UV light.
In today’s lab, you will add a small DNA molecule called a plasmid to a bacterium called E.
coli. These bacteria are the workhorse of modern recombinant DNA technology. The bacteria
you are starting with cannot produce GFP (since the gene is normally only found in the
jellyfish) and are killed by (aka “sensitive to”) the antibiotic ampicillin
The plasmid you will be adding is called “pGLO” and it contains two genes:
• Ampicillin Resistance – this is a gene that confers the dominant phenotype of resistance
to the antibiotic ampicillin.
• GFP – this is a gene that produces the GFP protein in bacteria.
You will add the pGLO DNA to bacteria that have been treated to make them “competent” –
that is, ready to take up DNA from the environment. However, only a very small fraction
(fewer than 1 in 1,000,000) of the bacteria will take up the DNA.
The bacteria that do take up the pGLO DNA now have the genes on pGLO added to their
genome. They have been “transformed”. These bacteria are now resistant to ampicillin and
produce GFP.
We next select for the transformed bacteria by growing the cells in the presence of ampicillin.
Un-transformed cells are sensitive to ampicillin and are killed. However, those that were
transformed with pGLO are resistant to ampicillin and will grow.
We will be growing our cells on solid medium, so a single surviving cell will give rise to a
“colony” of 108 cells – a small pile of cells, all descendants of that original transformed cell. All
the cells in the colony will carry pGLO and thus be ampicillin resistant and make GFP.
GFP II: Transformation - 3
The genetic map of pGLO is shown below:
You can see the genes for ampicillin resistance (AmpR) and GFP; each of these genes has a
promoter. We will talk more about plasmids and how they work in lecture.
Procedure I: Transformation
WARNINGS:
1. In general, the lab is unforgiving of mistakes like using the wrong solution or taking the
wrong amount. The construction folks at “This Old House”, say “Measure twice; cut
once.“ We’ll adapt this to “Check twice; pipette once”.
2. Although the E. coli strain we use is non-pathogenic (it is not known to cause disease in
healthy individuals), you should be careful with it. Always wear gloves, don’t eat or drink in
lab, and wash your hands thoroughly when you are all done.
3. Sterile Technique: Contamination is a big problem – it’s a dirty world we live in. You
should assume that all surfaces are crawling with nasty microbes. Never let any of the
sterile picks, pipettes, etc touch anything except the tube, plate, solution, or colony. If you
even think that you’ve touched something you shouldn’t, discard the loop, pipette, etc and
get a clean one.
GFP II: Transformation - 4
Note that while the figures show plastic transfer pipettes, we will be using pipetmen.
Transformation Kit—Quick Guide
-pGLO
+pGLO
1) Label one closed micro test tube +pGLO and another -pGLO.
1. Label one
closed micro test tube
Transformation
Label both tubes with your group’s
name.
Place-pGLO.
them in the Kit—Quick Guide
+pGLO
and another
1. Label one
closed
micro with
test tube
foam tube rack.
Label
both tubes
your
-pGLO
+pGLO
-pGLO
-pGLO
+pGLO
+pGLO
+pGLO
+pGLO
+pGLO
+pGLO
+pGLO
+pGLO
+pGLO
-pGLO
-pGLO
-pGLO
-pGLO
-pGLO
+pGLO
-pGLO
+pGLO
+pGLO
+pGLO
+pGLO
+pGLO
QUICK GUIDE
GUIDE
QUICK
GUIDE
QUICK
QUICK GUIDE
+pGLO
-pGLO
+pGLO
-pGLO
+pGLO
Transformation
Kit—Quick Guide
+pGLO
and another
-pGLO.
group’s
name.
Place them in the
Label both
tubes
with
your
tube rack.
1. group’s
Labelfoam
one
closed
micro
test
name.
Place
them
in tube
the
+pGLO
and
another
-pGLO.
2) Open the tubes and using
P1000
set
to
“0
2
5”,
250 µl
foam tube rack.
Transformation
Kit—Quick
Guide
Label
both
tubes
with
your
transfer 250μl of transformation solution (CaCl2) into
250 µl
group’s name. Place them in the
each tube.
1. Label foam
one closed
micro test tube
tube rack.
+pGLO and
another
2. Open
the -pGLO.
tubes and using a
250 µl
Label both tubes
with
yourpipet, transfer
sterile transfer
group’s
name.
Place
them
in
the
2. Open the
and using a solution
250tubes
µl of transformation
Transformation
foam sterile
tube rack.
transfer) pipet,
transfer
solution
(CaC1
into
each
tube.
2
250 µl of transformation solution
Transformation
250 µl
2. (CaC1
Open the
tubes and using a
solution
2) into each tube.
sterile transfer pipet, transfer
250 µl of transformation solution
Transformation
3. Place
the tubes on crushed ice. solution
(CaC1
2) into each tube.
2. Open the tubes and using a
Do not use cubed ice.
sterile
transfer
transfer
3. Place
thepipet,
tubes
on crushed ice.
250 µlDo
of not
transformation
solution
Transformation
use
cubed
3) Place the tubes on crushed
ice.each
Dotube.
not ice.
use cubed ice.
solution
(CaC12) into
Ice
4.
Use
a
sterile
loop
to
pick
up
3. Place the tubes on crushed ice.a
single
colony
of
bacteria
from
your
Do not
use cubed
ice.
Ice
4. Use
a starter
sterile
loop
pickup
upthe
a +pGLO
plate.toPick
single colony
of immerse
bacteria from
yourinto the
tube and
the loop
plate.on
Pick
up the
+pGLO
3. Placestarter
the tubes
crushed
ice.at the
solution
Ice
4. tube
Use and
atransformation
sterile
loop to
pick
upinto
a the
immerse
the
loopSpin
bottom
of
the
tube.
the loop
Do not
use
cubed
ice.
single
colony
of
bacteria
from
your
transformation
solution
at the
your
index
finger and
starterbetween
plate.
Pick
up
thethe
+pGLO
bottom
of
the until
tube.
Spin
loop is
thumb
the
entire
colony
tube andyour
immerse
the
loopand
into the
index
finger
Ice
4. Use abetween
sterile
loop
to
pick
up
a
dispersed
in the
transformation
transformation
solution
at theis
thumb
until
the
entire
colony
(withfrom
no floating
single colonysolution
of bacteria
your chunks).
bottom of in
thethe
tube.
Spin the loop
dispersed
transformation
4) Use a sterile loop to starter
pickup
a Place
single
colony
thethe
tube
backof
in the tube
plate.
Pick up
+pGLO
between
your
index
finger
and
solution
(with
no
floating
rack
inthe
the
ice.
Using
a new sterile
tube plate.
and
immerse
loop
intochunks).
the
bacteria from your starter
Pick
up
the
thumb
until
the
entire
colony
is
Place the
tube
back
inthe
the-pGLO
tube
loop,
repeat
for
transformation
solution
at
the
+pGLO tube and immerse rack
the
loop
thea new steriletube.
dispersed
ininto
the
transformation
in
the
ice.
Using
bottom of5.theExamine
tube. Spin
the
loopplasmid DNA
-pGLO
the
pGLO
solution
(with
floating
chunks).
transformation solution
at loop,
the
bottom
of
the
repeat
forno
the
-pGLO
tube.
between
your
index
finger
and
solution
with
the
UV
lamp.
Note
Place
the
tube
back
in
the
tube
tube. Spin the loop between
your
index
finger
thumb
until
the the
entire
colony
is Immerse
-pGLO
5. Examine
pGLO
plasmid
DNA
rack inyour
the observations.
ice.
Using
a new
sterilea new
and thumb until the entire
colony
is
dispersed
dispersed
in
the
transformation
solution
with loop
the
UV
lamp.
Note
sterile
into-pGLO
the plasmid
loop, repeat
for the
tube. DNA
solution
(with
no floating
chunks). a loopful.
in the transformation solution
(with
no floating
your
observations.
Immerse
new
stock
tube.
Withdraw
-pGLO
5. sterile
Examine
the
pGLO
plasmid
DNA
Place
the
tube
back
in
the
tube
loop
into
the
plasmid
DNA
chunks). Place the tube backsolution
in the
tube
rack
There
should
be ain
film of plasmid
with
the
UV
lamp.
Note
rack instock
the ice.
Using
a newathe
sterile
tube.
Withdraw
loopful.
solution
across
ring.
This is
the ice. Using a new sterile
loop,
repeat
for
the
- a new
your
observations.
Immerse
loop, repeat
for the to
-pGLO
tube.
There
should
be
a film
of
plasmid
similar
seeing
a
soapy
film
pGLO tube.
sterile loop
into the
the plasmid
solution
across
ThisDNA
is soap
-pGLO
aplasmid
ringring.
for DNA
blowing
5. Examine
theacross
pGLO
stock tube.
Withdraw
a loopful.
similar
to
a soapy
film into the cell
Mix
the
loopful
solution
withbubbles.
theseeing
UV lamp.
Note
There should
beblowing
a film ofsoap
plasmid
across
a ring Immerse
for
suspension
of the
tube.
your observations.
a +pGLO
new
solution
across
the
ring.
This
iscell
Mix
the loopful
intoµlthe
Optionally,
pipet
10
of pGLO
sterilebubbles.
loop into
the plasmid
DNA
similar to seeing
a+pGLO
soapy film
plasmid DNA
of the
tube.tube and
plasmid
into
the +pGLO
stock suspension
tube. Withdraw
a loopful.
-pGLO
across a ring
for10
blowing
soap
Optionally,
pipet
µl
of
pGLO
-pGLO tube and return
There shouldmix.
beClose
a filmthe
of plasmid
plasmid DNA
bubbles.into
Mixthe
the+pGLO
loopful tube
into the
cell
plasmid
and
-pGLO
it tothe
thering.
rack This
on ice.
add
solution
across
is Do not
suspension
of
the
+pGLO
tube.
Close
the
-pGLO
tube
returntube.
plasmid
DNA film
to
theand
-pGLO
similarmix.
to seeing
a soapy
Optionally,
pipet
10Do
µl of
pGLO
theWhy
rack
on ice.
not
not?
Close
the add
-pGLGO tube plasmid DNA
acrossit to
a ring
for blowing
soap
plasmidDNA
into the
+pGLO
tube
and
-pGLO
plasmid
to
the
-pGLO
tube.
to the
on ice.
bubbles. Mixand
thereturn
loopfulitinto
therack
cell
mix.
Close
the
-pGLO
tube
and
return
Why
not?
Close
the
-pGLGO
tube
suspension
of
the +pGLO
tube. on ice for
Incubate
the
tubes
it to6.return
the
rack
onthe
ice.rack
Do on
notice.
add
and
it10to
Optionally,
pipet
µl
of pGLO
10
min.
Make
sure to
push theplasmid DNA
plasmid DNA to the -pGLO
tube.
Rack
Ice
plasmid
intotubes
thethe
+pGLO
tube
and
6. Incubate
tubes
on
ice
for tube
-pGLO
all
the
down
in the
Why not?
Close
theway
-pGLGO
mix. Close
therack
-pGLO
tubebottom
and
return
10
min.
Make
sure
to
push
the
so
the
of
the
tubes
and return it to the rack on ice.
Rack
Ice
it to the
rackall
onthe
ice.
Do
not
addin contact
tubes
way
down
the
stick
out
and
make
with
18
GFP
II:
Transformation
6.
Incubate
tubes
on
icetubes
for - 5
plasmid
DNA
to
the
-pGLO
tube.
rack
sothe
thethe
bottom
of
the
ice.
10 min.
sure contact
to push
the
Why not?
Close
themake
-pGLGO
tubewith
stick
out Make
and
18
Rack
Ice
tubes
all
the
way
down
in
the
and return
it to the rack on ice.
the ice.
rack so the bottom of the tubes
6. Incubate the tubes on ice for
stick out and make contact with
18
10 min. Make sure to push the
the ice.
-pGLO
-pGLO
+pGLO
+pGLO
-pGLO
O
O
O
O
O
O
O
O
+pGLO
QUICK GUIDE
8. Heat shock. Using the foam rack
7. While
the tubes
are sitting
8. on
Heatthe
shock.
Using
the foam
rack
8) Heat
shock.
Using
rack
as aboth
holder,
as foam
a holder,
transfer
the (+)
ice,transfer
label yourboth
four agar
pGLO
pGLO
as pGLO
a holder,
transfer
both
the
(+)
pGLO
pGLO
pGLOand
and
(-)
pGLO
tubes
into
theplates
(+)
(-)
pGLO
tubes
LB/
LB/amp
ar a
B / a m ptubes into
/
on the bottom (not the lid) as
a
m
p
pGLOthe
andwater
(-)LpGLO
LB
bath,
set
at
42°C,
for
intoonthe
bath,
at bath,
42°C,
for
exactly
50
shown
thewater
diagram.
theset
water
at 42°C,
for sure
exactly
50set
seconds.
Make
to
seconds. Make sure
to push
the tubes
allway
the
exactly
50 seconds.
Make
sure
to down
push
the tubes
all the
way down in the rack
the
bottom
of
the
push so
the
tubes
all
the
way
down
in the rack so the bottom oftubes
the
8. Heat
shock.
Using
foaminrack
Water bath
the tubes
rack so
the out
bottom
of
the contact
stick
out
andthemake
contact
with
the
warm
stick
and
make
Water bath
as a holder, transfer both the
(+) with
tubes
stick
out
and
make
contact
the
warm
water.
When
the
water. When the 50 seconds have passed, place
pGLO and (-) pGLO tubes into
with the
warm
water.
When
the place
42°C for 50 sec
Ice
Ice
50
seconds
have
passed,
For
the
best
42°C for 50 sec
theboth
water tubes
bath, setback
at 42°C,on
for50ice.
Ice
Ice
seconds
haveback
passed,
place
both tubes
on ice.
For the
transformation
thetransformation
change
from
the the
ice
exactly
50 seconds. Makeresults,
sure
to tubes
both
back
on ice.
For
the
best
results,
push
the tubes
all theand
way down
(0°C)
to 42°C
then
back to
thethe
ice
best transformation
results,
the tobe rapid. Incubate tubes on ice for 2 min.
change
from
icemust
(0°C)
in the rack so the bottom of change
the 42°C
fromand
thethen
ice (0°C)
back to the ice
Water bath
tubes stick out and make contact
42°C and
then
the ice tubes
must
be back
rapid.toIncubate
with the warm water. Whenmust
the on
be ice
rapid.
tubes
for 2Incubate
min.tubes
250 µl
Remove
the rack
containing
the
from
the
42°C for
50 ice
sec and place
Iceon the bench top. Open the
50 9)
seconds
have passed,
place
on ice for 2 min. Ice
250 µl
both
tubes back
on ice.
For using
the9. Remove
rack containing
the 250μl of LB nutrient broth to the tube and
pGLO+
tube
and,
a newthe
sterile
pipet, add
9.
Remove
the
rack
containing
the
best
transformation
results,
the
tubes
from
the
ice
and
place
on pGLO- tube. Incubate the tubes for 30 min at
reclose it. Repeat with a new sterile pipet for the
frombench
the ice
and
place
on and,
change from the ice (0°C) totubes the
top.
Open
a tube
room temperature.the bench top. Open a tube and,
42°C and then back to the ice
using a new sterile pipet, add
using 250
a new
sterile
pipet, add
must be rapid. Incubate tubes
µl of
LB nutrient
broth to the
250 µltube
of LB
nutrient
broth
to 250
the µlwith
on ice for 2 min.
and
reclose
it. Repeat
tube and
reclose
it. Repeat
with
a new
sterile
pipet for
the other
LB-Broth
9. Remove the rack containing
the tube.
a new
sterile
pipet for
other
Incubate
thethe
tubes
for
LB-Broth
tubes from the ice and place
on Incubate
tube.
tubes
for
10 min atthe
room
temperature.
the bench top. Open a tube10
and,
min at room temperature.
using a new sterile pipet, add10. Gently flick the closed tubes with
100 µl
250 µl of LB nutrient broth
the your
10. to
Gently
flickfinger
the closed
tubes
with
to mix.
Using
a new
100
µl
tube and reclose it. Repeatyour
with finger
topipet
mix. Using
a new
sterile
for each
tube,
a new sterile pipet for the sterile
other pipet
pipet
forµleach
tube,
100
fromLB-Broth
each of the
tube. Incubate the tubes forpipet 100
tubes
theeach
corresponding
µl to
from
of the
10 min at room temperature.
as shown on the diagram
tubes plates,
to the corresponding
the
appropriate
plates.
plates,onto
as shown
on
diagram
GFP
II:the
Transformation
-6
10. Gently flick the closed tubes
with
+p
-p
-p
+p
onto
the appropriate plates.
GL
100 µl
GL
GL
GL
your finger to mix. Using a new
+ p L B / a m p + p LB/am
LB/
a- p
p
r
p
LB
a
m
a
/
p
L
G
L
L
G
L
G
G
sterile pipet for each tube,11. Use a new sterile loop for each
LB
LB/
LB/
/amp/ara
LB
amp
amp
pipet 100 µl from each11.
of the
Spreadloop
the suspensions
Use aplate.
new sterile
for each
tubes to the correspondingplate. evenly
the surface of the
Spreadaround
the suspensions
QUICK GUIDE
+pGLO
+pGLO
+pGLO
loop,
repeat
the tube
-pGLO tube.
Place the
tube
backfor
in the
rack5.
in the
ice. Using
a newplasmid
sterile DNA
-pGLO
Examine
the pGLO
loop, repeat
for the
solution
with-pGLO
the UVtube.
lamp. Note
your
Immerse
-pGLO
5. Examine
theobservations.
pGLO plasmid
DNA a new
sterile
loop
into
the
plasmid
solution with the UV lamp. Note DNA
5) Examine the pGLOyour
plasmid
DNA
solution
stock tube.
Withdraw
loopful.
observations.
Immerse
aanew
There
should
be a film
of plasmid
with the UV lamp. Note
observations.
sterileyour
loop
into
the plasmid
DNA
solution
across
ring. This is
Withdraw
a the
loopful.
Using a pipettor, put stock
10μltube.
of
pGLO
plasmid
similar
to
seeing
a
soapy
film
shouldClose
be a filmthe
of plasmid
into the +pGLO tube There
and mix.
+pGLO
across
a
ring
for
blowing
soap
solution across the ring. This is
tube and return it to the
rack
on ice.
Do
not
add
bubbles.
the loopful
similar to
seeing Mix
a soapy
film into the cell
plasmid DNA to the -pGLO
tube.
Why
not?
of the soap
+pGLO tube.
across asuspension
ring for blowing
Optionally,
pipet
10
µl
of
pGLO
Close the -pGLGO tube
and
return
it tointo
the
rack
bubbles.
Mix
the loopful
the
cell
plasmid DNA
plasmid
into
the
+pGLO
tube
and
-pGLO
suspension of the +pGLO tube.
on ice.
mix.pipet
Close10
theµl-pGLO
tube and return
Optionally,
of pGLO
plasmid DNA
itinto
to the
on ice.
Doand
not add
therack
+pGLO
tube
-pGLO
6) Incubate the tubes plasmid
on
ice
for
10
min.
Make
plasmid
DNA
to
the
-pGLO
tube.
mix. Close the -pGLO tube and return
sure to push the tubesit toallthethe
down
the tube
Why
not?
Close
thein
-pGLGO
rackway
on
ice.
Do not
add
and
return
toout
the and
rack
on
ice. contact
rack so the bottom ofplasmid
the tubes
stick
make
DNA
to
theit -pGLO
tube.
Incubate
the-pGLGO
tubes ontube
ice for
Why6.not?
Close the
with the ice.
10 min.
Make
to push the
and return
it to the
racksure
on ice.
Rack
Ice
tubes
all theon
way
6. Incubate
the tubes
icedown
for in the
rack
so
the
bottom
of
the
tubes
10 min. Make sure to push the
stick
make
contact with
Rack
Ice
18
tubes all
the out
wayand
down
in the
the
ice.
rack so the bottom of the tubes
stick out and make contact with
18
theWhile
ice. the tubes are sitting on
7.
7. While
the label
tubes
are
sitting
on plates
7) While the tubes
are sitting
on
ice,
label
ice,
your
four
agar
pGLO
pGLO
pGLO
pGLO
LB/
ice,
label
your
four
agar
plates
pGLO
pGLO
LB/amp
onthe
the bottom
(not
the lid) as
amp/ara pGLO LB/amp pGLO L B
your four agar plates on
bottom
(not
LB/
LB/amp
LB/amp
on theshown
bottomon
(not
lid) as
amp/ara
LB
thethe
diagram.
the lid) as shown on
the diagram.
shown on the diagram.
-pGLO
+pGLO
+pGLO
-pGLO
+pGLO
-pGLO
QUICK GUIDE
QUICK GUIDE
8. Heat shock. Using the foam rack
both tubes
ice.set
For
the back
wateron
bath,
atthe
42°C, for
a holder, transfer both the (+)
best transformation
results, Make
the as
exactly 50 seconds.
sure to
pGLO and (-) pGLO tubes into
change push
from the
the tubes
ice (0°C)
to
all the way down
water bath, set at 42°C, for
42°C and
thenrack
back
thebottom
ice the
in the
sotothe
of the
exactly 50 seconds. Make sure to Water bath
must betubes
rapid.
Incubate
tubes
stick out and make contact
push the tubes all the way
on ice flick
for
2 min.
µl
10) Gently
thewarm
closed
tubes
todown
mix.
Using a new sterile pipet for each
with
the
water.
Whenwith
the your finger 250
in the rack so the bottom
of the 42°C for 50 sec
Ice
Ice on the diagram
50
seconds
have
passed,
place
tube,
100ul
from
each of
toand
themake
corresponding
plates, as shown
Water bath
9. pipet
Remove
the rack
containing
thethe
tubestubes
stick out
contact
both
tubes
back
on
ice.
For
the
onto the
appropriate
plates.
sure
putwater.
the cells
tubes
from the ice and
place Be
on with
theto
warm
Whenon
the the jello-like agar medium, not the
besttop.
transformation
42°C for 50 sec
bench
Open a tuberesults,
and,50the
Ice
Ice
seconds have passed, place
plasticthe
lid!
change
from
the
ice
(0°C)
to
using a new sterile pipet, add both
tubes back on ice. For the
42°C
and thenbroth
backtotothe
the ice
250 µl of
LB nutrient
best transformation results, the
must
be
rapid.
Incubate
tubes
tube and reclose it. Repeat with change
from the ice (0°C) to
on
ice
for
2
min.
a new sterile pipet for the other42°C and then back
to the ice 250 µl
LB-Broth
tube.9.Incubate
mustthe
be rapid. Incubate tubes
Removethe
thetubes
rack for
containing
10 min at
room
temperature.
on ice
250 µl
tubes
from
the ice and place
onfor 2 min.
the bench top. Open a tube and,
9. Remove the rack containing the
10. Gently flick
the
closed
tubes
with
using
a new
sterile
pipet,
add
100
µl place on
tubes from the ice
and
your finger
to
mix.
Using
a
new
250 µl of LB nutrient broth to the
the bench top. Open a tube and,
sterile pipet
for reclose
each tube,
tube and
it. Repeat with
using a new sterile pipet, add
pipet 100
µl
from
each
of
the
a new sterile pipet for the other
LB-Broth
250 µl of LB nutrient broth
to the
tubes totube.
the corresponding
Incubate the tubes for
tube
and
reclose
it.
Repeat
with
plates, as
shown
on
the
diagram
10 min at room temperature.
a new sterile pipet for the other
onto the appropriate plates.
LB-Broth
+ p tubes for + p
-p
-p
tube. Incubate the
GL
10. Gently flick the closed tubes with
L
GL
L
G
G
LB/
LB/
10
min
at
room
temperature.
p 100 µl LB/amp/ara
p
LB
m
a
m
a
your finger to mix. Using a new
11. Use a new sterile loop for each
sterile pipet for each tube,
plate. Spread the suspensions
10. Gently flick the closed tubes with
pipet 100 µl from each of the
100 µl
evenly around the surface of the your finger to mix. Using a new
tubes to the corresponding
agar by quickly skating the flat sterile pipet for each tube,
plates, as shown on the diagram
surface of a new sterile loop
pipet 100 µl from each of the
onto the appropriate plates.
back and forth across the plate tubes to the corresponding
+p
-p
-p
+p
GL
GL
GL
GL
surface.
plates, as shown on theLdiagram
LB
LB/
B/amp
p
/amp/ara
LB
am
ontoeach
the
appropriate plates.
11) Use a11.
new
loop loop
for each
plate.
Usesterile
a new sterile
for
+p
-p
+p
GL
plate.
theevenly
suspensions
12. Stack
yourSpread
plates and
tape around the
GL
GL
Spread
the up
suspensions
LB
LB/
LB/
a
p
r
/
m
a
amp/a
amp
evenly
around
the
surface
of
the
them
together.
Put
your
group
surface of the agar by quickly
skating
the
11.
Use
a
new
sterile
loop
for
each
agar
by period
quicklyon
skating
name and
class
the the flat
plate.
Spread
flat surface
of
a new
loop
back
andthe suspensions
surface
of asterile
newplace
sterile
loop
bottom of
the stack
and
the
evenly
around
the surface of the
forth across
the plate
surface.
back
and
forth
the plate
stack upside
down
in across
the 37°C
agar
by
quickly
skating the flat
surface.
incubator
until the next day.
surface of a new sterile loop
back and forth across the plate
12. Stack up your plates andsurface.
tape
them together. Put your group
19
class period
the them together. Put your group name and
12) Stack upname
yourand
plates
and12.on
tape
Stackthe
up your plates and tape
bottom of the stack and place
class periodstack
on the
bottom
of
the
stack
andPut
place
stack upside down in the
them
together.
your the
group
upside down in the 37°C
name
and
class
period
on
the
37°C incubator
untiluntil
thethenext
incubator
next day.
day.
bottom of the stack and place the
stack upside down in the 37°C
incubator until the next day.
O
O
O
O
O
GFP II: Transformation - 7
O
O
O
O
Lab Report
There is no lab report for this session.
O
O
19
Once your cells have grown, your TA will put them in the refrigerator so you
can look at them during the last week of lab.
19
-p
GL
LB
IGV Practice
Many students find it challenging to use the Integrative Genome Viewer (IGV) on the SPOC.
In this part of the lab, your TA will help you to learn how to use the IGV by working through
some of the SPOC IGV problems.
GFP II: Transformation - 8