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             <p>o/n culture <br/> of E. coli <br/> TOP10</p>
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             <p>o/n culture <br/> of <i>E. coli</i> <br/> TOP10</p>
 
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             <p>o/n culture <br/> of E. coli <br/> BL21(DE3)</p>
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             <p>o/n culture <br/> of <i>E. coli</i> <br/> BL21(DE3)</p>
 
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             <p>PCR purification <br/> for VioA <br/> 2nd PCR</p>
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             <p>PCR purification <br/> for BglX <br/> 2nd PCR</p>
 
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             <p>GFP <br/>expression</p>
 
             <p>GFP <br/>expression</p>
 
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<p style="font-size:16px">Click <a href="https://static.igem.org/mediawiki/2015/f/fc/Aalto-Helsinki_week18.pdf" target="_blank">here</a> to read the full lab notes of week 18.</p></div>
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Latest revision as of 04:29, 29 October 2015

Lab Book

Before reading our lab notes, familiarize yourelf with our excel sheet, which specifies the nomenclature we've used in out project. You can also find all of our primers on this excel.

Week 1

Worked With

WorkFlow

May 18th -
May 22nd

Preparations
Plates

Making Cm
plates

Stored in
+4°C

Competent Cells

Preparing
solutions for
competent cells

Stored in
+4°C

o/n culture
of E. coli
TOP10

Chemical
competent
cells protocol

Transformation
efficiency kit

o/n culture
of E. coli
BL21(DE3)

Chemical
competent
cells protocol

Transformation
efficiency kit

Colonies have
the wrong
color

Propagations

Transformed
  AH001-AH008
into TOP10

 AH001-AH004
& AH006

Stored in
+4°C

No colonies for
  AH005, AH007
or AH008

Click here to read the full lab notes of week 1.

Week 2

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

Re-transform
AH005
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

 Transform
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

 Transform
into TOP10

Plates in
rt over
weekend

AH001+
   AH006+AH008
-> AH011

Repeat
ligation

Restrict &
extract
from gel

 Transform
into TOP10

Plates in
rt over
weekend

Click here to read the full lab notes of week 2.

Week 3

Worked With

WorkFlow

June 1st -
June 5th

Preparations
Propagating

Transform
    AH020 & AH021
into TOP10

Plates in rt
 over weekend

Testing RBS's
Assembly

No colonies
for AH011
or AH014

re-assemble

Restrict &
extract
from gel

Ligate &
transform
into TOP10

No colonies

AH005 &
AH013 OK

o/n cultures

MiniPrep

Assembling
AH015

  Ligate AH002+
  AH005+AH008

Restrict &
extract
from gel

Transform into TOP10

o/n cultures

No colonies
on the plate

Click here to read the full lab notes of week 3.

Week 4

Worked With

WorkFlow

June 8th -
June 12th

Preparations
Propagating

o/n cultures
for AH020
& AH021

MiniPrep

Testing RBS's
Assembly

Re-assembling
  AH011, AH013
& AH014

Transform into TOP10

o/n cultures

MinPrep

Assembling
AH015

  Ligate AH002+
  AH005+AH008

Restrict &
extract
from gel

Transform into TOP10

o/n cultures

MiniPrep

Click here to read the full lab notes of week 4.

Week 5

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 AH011
& AH013-AH015
by gel
electrophoresis

AH011, 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

Click here to read the full lab notes of week 5.

Week 6

Worked With

WorkFlow

June 22nd -
June 26th

Preparations
Linearized Backbones

o/n cultures of
AH007-AH009

MiniPrep

Restrict &
extract
from gel

Testing RBS's
Assembly

Re-assembling AH016 &
AH018

Transform
into
BL21(DE3)

No colonies

Propagation

o/n cultures of
 AH016 & AH018
in TOP10

MiniPrep

Transform
into
BL21(DE3)

Plates in rt
over the
weekend

Measurement

AH018 in
BL21(DE3)

o/n culture

IPTG induction &
    blue chromoprotein
measurement

No blue chromoprotein

Refresh on
IPTG plate

No blue chromoprotein

Click here to read the full lab notes of week 6.
Blue chromoprotein measurement data from 23.6. can be found here.

Week 7

Worked With

WorkFlow

June 29th -
July 3rd

Preparations
Linearized Backbones

Transform
AH007-AH009
into TOP10

No colonies
(mistake in
protocol)

Use iGEM's
linearized
backbones

Testing RBS's

o/n cultures of
  AH016&AH018
in BL21

IPTG
induction

No Blue
chromoprotein

Refresh AH016 &
AH018 on
IPTG plate

No blue
chromoprotein

Violacein Plasmid
Construction

PCR AH020
with
P012&P013

PCR
purification

VioA waits
for 2nd PCR

PCR AH20
with
P024&P025

PCR
unsuccesful

PCR AH20
with
P034&P035

PCR
purification

VioE waits
for 2nd PCR

Click here to read the full lab notes of week 7.
Blue chromoprotein measurement data from 30.6. can be found here.

Week 8

Worked With

WorkFlow

July 6th -
July 10th

Preparations Competent Cells

Preparing
  PK BL21(DE3)
competent cells

(Cells
called PK's
BL21(DE3))

Cells stored
in -80°C

Cellulose Plasmid
Construction

PCR AH021
with
P019&P020

Problem
with PCR
machine

Re-do PCR

PCR
purifcation

BglX waits for
2nd PCR

AH029 into
TOP10

Transformants
stored in
+4°C

Testing RBS's

Transform AH016
& AH018 into
PK BL21(DE3)

Refresh
transformants
on IPTG plates

No blue
chromoprotein

Violacein Plasmid
Construction

Gradient PCR for
AH020 with
P024&P025

PCR
purification

Click here to read the full lab notes of week 8.

Week 9

Worked With

WorkFlow

July 13th -
July 17th

Preparations
Propagations

o/n culture
of AH029

MiniPrep

CAR2 OK

PCR
purification

Low yield

Propane Plasmid 1 Construction

Construct
from 3 parts:
CAR1-3

PCR for
CAR1-3

CAR1 & 3
unspecific bands

Gradient PCR
for CAR3

PCR with
DMSO
for CAR3

Store
in -20°C

PCR with DMSO
for CAR1

No product

PCR with
DMSO for
AtoB1

Store
in -20°C

Propane Plasmid 2 Construction

Construct from
4 parts:
AtoB1-4

PCR for
AtoB 1-4

Gradient PCR
for AtoB2

PCR with
DMSO for
AtoB2

Store
in -20°C

Gradient PCR
for AtoB3

Extract &
purify from gel

Store
in -20°C

Extract &
purify AtoB4
from gel

Store
in -20°C

GFP Brick Construction

o/n culture
of AH044

MiniPrep

Click here to read the full lab notes of week 9.

Week 10

Worked With

WorkFlow

July 20th -
July 24th

Preparations Linearized Backbones

Transform AH043 into TOP10

o/n culture

MiniPrep

Restrict

Propane Plasmid 1 Construction

Gradient PCR
for CAR1

Extract &
purify from gel

Low yield

TOPO Blunt End
cloning and
transform into TOP10t

Screen with
colony PCR

Screen more
colonies

CAR2 PCR
ran on gel

Gradient PCR

Extract &
purify
from gel

Illegal restriction
site found
in the part

CAR3 PCR
with DMSO
run on gel

Extract
purify from gel
unclean product

TOPO Blunt End
cloning and
transform into TOP10t

Screen with
colony PCR

Extract &
purify
from gel

Propane Plasmid 2 Construction

Run AtoB1
PCR with
DMSO on gel

Extract &
purify from gel

Low yield

Run AtoB2
PCR with
DMSO on gel

Extract &
purify from gel

Low yield

Click here to read the full lab notes of week 10.

Week 11

Worked With

WorkFlow

July 27th -
July 31st

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 1 Construction

Continue
CAR1 colony
screening

Screen more
colonies

PCR
purification

PCR for
new CAR2

Gradient PCR
with KAPA
polymerase

PCR
purification

Re-do colony
PCR for CAR3
successful TOPO colony

Re-do
colony PCR

o/n culture of
the colony

MiniPrep

PCR

KAPA
gradient PCR
for CAR3

Gel purification

Propane Plasmid 2 Construction

TOPO Blunt End
Cloning for AtoB1
and transformation
into TOP10

Screening
with colony
PCR

PCR
purification

KAPA gradient
PCR for AtoB3&4

PCR purification
for AtoB3

Gel
purification
for AtoB4

Cellulose Plasmid Construction

2nd PCR
for BglX
(from AH021)

Stored in -4°C
over the
weekend

PCR for
Cen1 & Cen2

Gradient PCR
with KAPA
polymerase

PCR
purification for
Cen2

Gel purification
for Cen1

Violacein Plasmid Construction

2nd PCR
for VioA
(from AH020)

Stored in -4°C
over the
weekend

Click here to read the full lab notes of week 11.

Week 12

Worked With

WorkFlow

August 3rd -
August 8th

Preparations Linearized Backbones

Check
restricted
AH043 on gel

Use for CAR
OE-PCR

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

Fixing Commercial Backbones

Restrict and
ligate AH006
into AH031&AH033

Transform into TOP10

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

Violacein Plasmid Construction

PCR purification
for VioA
2nd PCR

Stored
in -20°C

Cellulose Plasmid Construction

PCR purification
for BglX
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

Propane Measurement

o/n culture
of Pauli's
propane strain

Propane measurement

No propane
detected

Click here to read the full lab notes of week 12.

Week 13

Worked With

WorkFlow

August 10th -
August 14th

Amphiphilic Protein Construction

PCR amph
with P044&P045

No product

Gradient
PCR

No product

Continue with
last week's
product

Amph with Linker Validation

Colony PCR
amph with
P045&P046

O/n culture

MiniPrep

Restrict &
check
on gel

Extract & purify
Amph P046&P051
and P045&P046
from gel

Assemble both
into pSB1C3
backbone

Transform
into TOP10

Amph no term Validation

Colony PCR
amph with
P046&P051

O/n culture

MiniPrep

Restrict &
check
on gel

Preparations Backbones

Restrict AH043

Stored
in -20°C

GFP & Amph Bricks
Assembly

Restrict &
ligate GFP and
Amph into AH009

Transform
into TOP10

Screen with
colony PCR

o/n culture

MiniPrep

Check on
gel

Send for
sequencing

Propane Plasmid 2 Assembly

NEBuilder
protocol

Transform
into TOP10

Screen with
colony PCR

Re-do NEBuilder
and transfrom
to TOP10

Screen with
colony PCR

o/n culture of
one potential
colony

o/n culture
turned red

ELIC protocol

Transform
into TOP10

No colonies

Cellulose Plasmid Assembly

NEBuilder protocol
and transform
into TOP10

Screen with
colony PCR

Mistake in
protocol

Re-do NEBuilder
and transform
into TOP10

Screen with
colony PCR

o/n cultures and
MiniPrep of
white colonies

Restrict &
check on gel

ELIC
protocol

Transform
into TOP10

Screen with
colony PCR

Mistake in
protocol

Re-do ELIC
and transform
into TOP10

Screen with
colony PCR

GFP Brick Validation

Restrict AH013,
AH015 & AH007

Ligate AH007,
AH013 & GFP

Transform
into TOP10

RT over
weekend

Ligate AH007,
AH015 & GFP

Transform
into TOP10

RT over
weekend

Click here to read the full lab notes of week 13.

Week 14

Worked With

WorkFlow

August 17th -
August 21st

Preparations
Propagations

Transform
AH045 into
TOP10

o/n culture

MiniPrep

Propane Plasmid 1 Assembly

Restrict &
ligate CAR
into AH009

Transform into TOP10

o/n culture

MiniPrep

Restrict &
check on gel

Sequencing of
Propane Plasmid 1
unsuccessful

Send construct in AH009 for sequencing

Validation

Transform into
Pauli's
BL21(DE3)

Amphiphilic Propane 1

Gradient PCR
for CAR amph
parts

CAR amph3
purified
from gel

PCR purification
for CAR
amph4

Propane Plasmid 2
Construction

OE-PCR

No product

Check old
NEBuilders
on gel

Re-transform
into TOP10

o/n cultures of
colorless
colonies

MiniPrep

Restrict &
check
on gel

Cellulose Plasmid Construction

OE-PCR

Extract &
purify
from gel

Concentration too low

PCR
purification

NEBuilder with AH009

Transform into
NEB5alpha
over weekend

Check old
NEBuilder
on gel

Re-transform into TOP10

o/n of colorless
colonies

MiniPrep

Restrict &
check
on gel

GFP Assembly

2A assembly for
AH013/AH015,
GFP & pSB1C3

Transform
into TOP10

Colonies can’t
be separated

3A assembly
into pSB1T3

Transform
into TOP10

No colonies

Propagate GFP in AH009

MiniPrep

Send to Slovenia

Click here to read the full lab notes of week 14.

Week 15

Worked With

WorkFlow

August 24th -
August 28th

Propane Plasmid 1
Validation

Prepare
SDS-PAGE
samples

Unclear
results

Analytical
restriction

Wrong sized
bands

Prepared
cells for
purpald test

Stored
in -70°C

Propane Plasmid 2
Construction

Repeat
NEBuilder

Transform into TOP10

Repeat
NEBuilder
with AH009

Transform into TOP10

Colony PCR

GFP Biobrick Assembly

3A Assemble
GFP + AH045
in pSB1K3

Transform
into TOP10

O/n cultues

MiniPrep

Restrict &
Check
on gel

2A Assembly
GFP + AH045 in pSB1K3

Transform
into TOP10

Store in +4 °C

GFP+Amph Assembly

3A Assemble
AH045-GFP +
amphlinker in pSB1A3

Transform
into TOP10
over weekend

Cellulose Plasmid Construction

Repeat
NEBuilder

Transform into TOP10

Repeat
NEBuilder
with AH009

Transform into TOP10

Colony PCR

Amphiphilic Propane1

NEBuilder

Transform
into TOP10

Colony PCR

O/n culture

MiniPrep

Click here to read the full lab notes of week 15.

Week 16

Worked With

WorkFlow

August 31st -
September 4th

Propane Plasmid 1
Validation

New
SDS-PAGE

Unclear
results

Propane Plasmid 2
Construction

o/n culture
of NEBuilder
colonies

MiniPrep

Restrict &
check
on gel

No insert

Screen more
colonies with
colony PCR

Cellulose Plasmid Construction

o/n culture
of NEBuilder
colonies

MiniPrep

Restrict &
check
on gel

No insert

Screen more
colonies with
colony PCR

Amph Propane 1 Construction

Restrict &
check
on gel

Re-do
NEBuilder

Transform
into
NEB5alpha

O/n cultures

MiniPrep

GFP Amph Assembly

O/n cultures

MiniPrep

Restrict &
check
on gel

O/n cultures
of more
colonies

MiniPrep, restrict
& check
on gel

Prepare samples
for microscopy

Problem with
imaging

Amphiphilic Brick Assembly

PCR purification

3A assemble
AH045 + Amph
in pSB1C3

Transform
into TOP10

O/n cultures & MiniPrep

Restrict &
Check
on gel

More o/n cultures & MiniPrep

Restrict &
Check on gel,
results unclear

Click here to read the full lab notes of week 16.

Week 17

Worked With

WorkFlow

September 7th -
September 11th

Propane Plasmid 1
Validation

Colony PCR
with
P001 & VR

Results
next week

Propane Plasmid 2
Construction

o/n culture
of NEBuilder
colonies

MiniPrep

Restrict &
check
on gel

AtoB maybe
OK

Restrict &
check on
gel again

Unclear results

Colony PCR,
results
next week

Cellulose Plasmid Construction

o/n culture
of NEBuilder
colonies

MiniPrep

Restrict &
check
on gel

No insert

Due to the time
constraint, the
construction is
not repeated

Amph Propane 1 Construction

Restrict &
check
on gel

Incorrect size

Due to the time
constraint, the
construction is
not repeated

GFP Amph Assembly

Restrict &
check on gel

Incorrect size

Re-assemble

Transform
into TOP10

GFP not
expressed

Transform old
ligation mix
into TOP10

GFP not
expressed

Validation

Imaged with
fluorescent
microscope

O/w culture
for microscopy

Amphiphilic Brick Assembly

Restrict &
check on gel

Incorrect size

Re-assemble

Transform
into TOP10

O/n cultures

MiniPrep

O/w culture
for microscopy

Click here to read the full lab notes of week 17.

Week 18

Worked With

WorkFlow

September 14th -
September 18th

Propane Plasmid 1
Validation

Checking
colony PCR
results

Propane Plasmid 2
Construction

Checking
colony PCR
results

GFP Validation

Assembling
AH046

Restriction
and ligation

Transform
into TOP10

No blue
colonies

Re-assembling

Transform
into TOP10

No blue
colonies

Assembling
AH047

Restriction
and ligation

Transform
into TOP10

No blue
colonies

GFP & Amph Validation

Prepare samples
for confocal
microscope

Pictures
taken by
VTT

Amphiphilic
structures
not detected

GFP
expression

Amph Brick
Validation

Prepare samples
for confocal
microscope

Pictures
taken by
VTT

Amphiphilic
structures
not detected

Click here to read the full lab notes of week 18.