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Revision as of 07:59, 19 August 2015

Lab Book

Construct
worked with

Things done

Lab 1.1.2015
Monday

CAR

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Here is my
text, there
is lot of it

another CAR?

Here is my text

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Here is my text

Here is my text

Here is my text

Here is my
text, there
is lot of it

Ado?

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Here is my text

Ado cont.

Here is my text

Here is my text

Here is my text

Here is my text

Here is my
text, there
is lot of it

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

Tranformation
efficiency kit

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

Chemical
competent
cells protocol

Tranformation
efficiency kit

Colonies are
the wrong
color

Propagations

Transformed
  AH001-AH008
into TOP10

 AH001-AH004
& AH006

Stored in
+4°C

No colonies for
  AH005, AH007
or AH008

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

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

 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

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 &
tranform
into TOP10

No colonies

AH005 &
AH013 OK

o/n cultures

MiniPrep

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

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 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

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

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 TOP10

IPTG
induction

No Blue
chromoprotein

Refresh AH016 &
AH018 on
IPTG plate

No blue
chromoprotein

Violacein Plasmid
Construction

PCR AH20
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

Week 8

Worked With

WorkFlow

July 6th -
July 10th

Preparations Competent Cells

Preparing
  PK BL21(DE3)
competent cells

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

Week 9

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

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

Propane Plasmid 2 Construction

TOPO Blunt End
Cloning for AtoB1
and transformation
into TOP10

Screening
with colony
PCR

PCR
purification

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

KAPA gradient
PCR for AtoB3,4
& CAR3

Gel purification
for AtoB4
& Car3

PCR
purification
for AtoB3

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 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

Propane Measurement

o/n culture
of Pauli's
propane strain

Propane measurement

Results
available
next week

Week 13

Week 14

Week 15

Week 16

Week 17

Week 18