Worked With |
WorkFlow |
|
|
|
|
|
May 18th -
May 22nd |
Preparations
Plates |
Making Cm
plates
|
Stored in
+4°C
|
Preparations
Competent Cells |
Preparing
solutions for
competent cells
|
Stored in
+4°C
|
|
o/n culture
of E. coli
TOP10
|
Chemical
competent
cells protocol
|
Tranformation
efficiency kit
|
|
o/n culture
of E. coli
BL21(DE3)
|
Chemical
competent
cells protocol
|
Tranformation
efficiency kit
|
Colonies are
the wrong color
|
Preparations
Propagations |
Transformed
AH001-AH008
into TOP10
|
AH001-AH004
& AH006
|
Stored in
+4°C
|
|
|
No colonies for
AH005, AH007
or AH008
|
Worked With |
WorkFlow |
|
|
|
|
|
May 25th -
May 29th |
Preparations
Propagating |
Transformed
AH007-AH009,
AH005 & AH019
into TOP10
|
AH007-AH009
& AH019
|
o/n cultures
|
MiniPrep
|
|
o/n culture of
AH001-AH004
& AH006
|
MiniPrep
|
|
|
No colonies
for AH005
|
Re-transform
into TOP10
|
Plates in rt
over weekend
|
Checking
Competent Cells |
o/n culture
of BL21(DE3)
transformants
|
MiniPrep
|
PCR with
VF2 & VR
|
Smeared gels
|
|
|
|
AH001+
AH003+AH008
-> AH013
|
Repeat
ligation
|
Restrict &
extract
from gel
|
Transformation
into TOP10
|
Plates in rt
over weekend
|
Testing RBS's
Assembly |
Restrict
AH001-AH004
& AH006-AH008
|
Ligation
|
AH002+
AH004+AH008
-> AH014
|
Repeat
ligation
|
Restrict &
extract
from gel
|
Transformation
into TOP10
|
Plates in rt
over weekend
|
|
|
|
AH001+
AH006+AH008
-> AH011
|
Repeat
ligation
|
Restrict &
extract
from gel
|
Transformation
into TOP10
|
Plates in rt
over weekend
|
Worked With |
WorkFlow |
|
|
|
|
|
June 15th -
June 19th |
|
|
|
| |
|
AH016
wrong size
on gel
|
|
|
|
|
Colonies in
BL21(DE3)
|
o/n cultures
& MiniPrep
|
AH018 OK
on gel
|
Testing RBS's
Assembly |
Screening AH009
& AH013-AH015
by gel
electrophoresis
|
AH009, AH013
& AH015 OK
|
Assembling
AH016 &
AH018
|
Transform into TOP10 & BL21(DE3)
|
T7 promoter
doesn't work
in TOP10...
|
|
|
AH014
incorrect size
|
Assembling AH017
with multiple
AH014 plasmids
|
Restrict &
extract
from gel
|
Transform into BL21(DE3)
|
Colonies are
not blue
|
|
Re-assembling
AH016 &
AH018
|
Restrict
extract
from gel
|
Transform into
BL21(DE3)
|
Colonies are
not blue
|
Preparations
Linearized Backbones |
o/n cultures of
AH007-AH009
|
MiniPrep
|
Restrict &
extract
from gel
|
Low
concentration
|
Worked With |
WorkFlow |
|
|
|
|
|
June 22nd -
June 26th |
Testing RBS's
Assembly |
Re-assembling AH016 &
AH018
|
Transform
into
BL21(DE3)
|
No colonies
|
Testing RBS's
Propagation |
o/n cultures of
AH016 & AH018
in TOP10
|
MiniPrep
|
Transform
into
BL21(DE3)
|
Plates in rt
over the
weekend
|
Testing RBS's
Measurement |
AH018 in
BL21(DE3)
|
o/n culture
|
IPTG induction &
blue chromoprotein measurement
|
No blue chromoprotein
|
|
|
Refresh on
IPTG plate
|
No blue chromoprotein
|
Preparations
Linearized Backbones |
o/n cultures of
AH007-AH009
|
MiniPrep
|
Restrict &
extract
from gel
|
Worked With |
WorkFlow |
|
|
|
|
|
July 13th -
July 17th |
Preparations
Propagations |
o/n culture
of AH029
|
MiniPrep
|
|
|
CAR2 OK
|
PCR
purification
|
Propane Plasmids 1&2 Construction |
PCR for
CAR1-3 &
AtoB1-2
|
CAR1,3 &
AtoB1-2
unspecific bands
|
Gradient PCR
for CAR3 & 4
|
|
|
PCR with DMSO
for CAR1, 3
& AtoB1, 2
|
dunnowhat
|
GFP Brick Construction |
o/n culture
of AH044
|
MiniPrep
|
Propane Plasmid 2 Construction |
PCR for
AtoB3-4
|
Unspecific
bands
|
AtoB 4
extracted
from gel
|
Low
concentration
|
|
|
Gradient
PCR for
AtoB3
|
Extract &
purify
from gel
|
Worked With |
WorkFlow |
|
|
|
|
|
July 27th -
July 31st |
Propane Plasmid 1 Construction |
Continue CAR1
colony
screening
|
Screen more
colonies
|
PCR purification
|
|
PCR for
new CAR2
|
Gradient PCR
with KAPA
polymerase
|
PCR
purification
|
Preparations Linearized Backbones |
Re-restrict
AH043
|
Gel purification
|
Low yield
|
Re-restrict
|
Propagations |
o/n culture
of AH031
|
MiniPrep
|
|
Transform
AH033 into
TOP10
|
o/n culture
|
MiniPrep
|
Low yield
|
o/n culture
|
MiniPrep
|
Low yield,
but stored
in -20°C
|
Propane Plasmid 2 Construction |
TOPO Blunt End
Cloning for AtoB1
and transformation
into TOP10
|
Screening
with colony
PCR
|
PCR
purification
|
Cellulose Plasmid Construction |
PCR for
Cen1 & Cen2
|
Gradient PCR
with KAPA
polymerase
|
PCR
purification for
Cen2
|
|
|
|
Gel purification
for Cen1
|
|
2nd PCR
for BglX
(from AH021)
|
Stored in -4°C
over the
weekend
|
Propane Plasmid 1 Construction |
Re-do colony
PCR for CAR3
successful TOPO colony
|
Re-do
colony PCR
|
|
|
o/n culture of
the colony
|
MiniPrep
|
PCR
|
Violacein Plasmid Construction |
2nd PCR
for VioA
(from AH020)
|
Stored in -4°C
over the
weekend
|
|
KAPA gradient
PCR for AtoB3,4
& CAR3
|
Gel purification
for AtoB4 & Car3
|
|
|
PCR
purification
for AtoB3
|
Worked With |
WorkFlow |
|
|
|
|
|
August 3rd -
August 8th |
Propane Plasmid 1 Construction |
OE-PCR
without
backbone
|
Weak product
band
|
PCR purification
|
Gradient PCR
|
No product
|
|
OE-PCR with
backbone
|
Transform into TOP10
|
Red colonies
|
|
NEBuilder
protocol
|
Transform into TOP10
|
Screen with
colony PCR
|
o/n culture
|
MiniPrep
|
Send for
sequencing
|
Preparations Linearized Backbones |
Check
restricted
AH043 on gel
|
Use for CAR
OE-PCR
|
Low yield
|
|
Re-restrict
with high
amount of DNA
|
Extract &
purify
from gel
|
Low yield
|
|
New o/n culture
of AH043 (needed
more backbone)
|
MiniPrep
|
Remove insert
|
Use for CAR
NEBuilder
|
Violacein PlasmidConstruction |
PCR purification
for VioA
2nd PCR
|
Stored in -20°C
|
Cellulose Plasmid Construction |
PCR purification
for VioA
2nd PCR
|
Stored in -20°C
|
Amph protein |
Gradient PCR
P044&P045, P046&P051,
P045&P046
|
Extract &
purify from gel
P044&P045
|
DNA not clean
|
Store in -20°C
|
|
|
Stored P046&P051
and P045&P046
product gels
to +4°C
|
GFP protein |
Gradient PCR
with P028&P029
|
PCR purification
|
Preparations |
Restrict and
ligate AH006
into AH031&AH033
|
Transform into TOP10
|
Propane Measurement |
o/n culture
of Pauli's
propane strain
|
Propane measurement
|
Results
available
next week
|
