Difference between revisions of "Team:Aalto-Helsinki/LabBook"

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             <p>Use for CAR <br/>OE-PCR</p>
 
             <p>Use for CAR <br/>OE-PCR</p>
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            <p>Low yield</p>
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Revision as of 11:17, 18 August 2015

Lab Book

Construct
worked with

Things done

Lab 1.1.2015
Monday

CAR

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text, there
is lot of it

another CAR?

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Here is my text

Here is my
text, there
is lot of it

Ado?

Here is my text

Here is my text

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Here is my text

Ado cont.

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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

Week 1

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

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

 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

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

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

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

Testing RBS's
Measurement

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

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

Week 12

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

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

Week 13

Week 14

Week 15

Week 16

Week 17

Week 18