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

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<h2 id="week12">Week 12</h2>
 
<h2 id="week12">Week 12</h2>
 +
 +
<table >
 +
  <tr>
 +
    <td class="bottomline rightline first"><h3 style="padding-top:0;margin-top:0;margin-bottom:0;padding-bottom:0;">Worked With</h3></td>
 +
    <td class="bottomline"><h3>WorkFlow</h3></td>
 +
    <td class="bottomline"></td>
 +
    <td class="bottomline"></td>
 +
    <td class="bottomline"></td>
 +
    <td class="bottomline"></td>
 +
    <td class="bottomline"></td>
 +
    <td class="bottomline"><h3>August 3rd -<br/> August 8th</h3></td>
 +
  </tr>
 +
  <tr>
 +
        <td class="first rightline"><div class="heightdiv"><h3>Propane Plasmid 1 <span style="font-size:20px">Construction</span></h3></div></td>
 +
        <td class="white arrow" >
 +
            <p>OE-PCR <br/> without <br/> backbone</p>
 +
        </td>
 +
        <td class="blue arrow" >
 +
            <p>Weak product <br/>band</p>
 +
        </td>
 +
        <td class="white arrow" >
 +
            <p>PCR purification</p>
 +
        </td>
 +
        <td class="white arrow" >
 +
            <p>Gradient PCR</p>
 +
        </td>
 +
        <td class="red arrow" >
 +
            <p>No product</p>
 +
        </td>
 +
  </tr>
 +
  <tr>
 +
        <td class="first rightline"><div class="heightdiv"><h3></h3></div></td>
 +
        <td class="white arrow" >
 +
            <p>OE-PCR with <br/> backbone</p>
 +
        </td>
 +
        <td class="white arrow" >
 +
            <p>Transform into TOP10</p>
 +
        <td class="red arrow" >
 +
            <p>Red colonies</p>
 +
        </td>
 +
  </tr>
 +
  <tr>
 +
        <td class="first rightline"><div class="heightdiv bottomline"><h3></h3></div></td>
 +
        <td class="white arrow" >
 +
            <p>NEBuilder <br/> protocol</p>
 +
        </td>
 +
        <td class="white arrow" >
 +
            <p>Transform into TOP10</p>
 +
        <td class="blue arrow" >
 +
            <p>Screen with <br/>colony PCR</p>
 +
        </td>
 +
        <td class="white arrow" >
 +
            <p>o/n culture</p>
 +
        </td>
 +
        <td class="blue arrow" >
 +
            <p>MiniPrep</p>
 +
        </td>
 +
        <td class="white arrow" >
 +
            <p>Send for <br/>sequencing</p>
 +
        </td>
 +
  </tr>
 +
  <tr>
 +
        <td class="first rightline"><div class="heightdiv"><h3>Preparations <span style="font-size:20px">Linearized Backbones</span></h3></div></td>
 +
        <td class="blue arrow" >
 +
            <p>Check <br/>restricted <br/>AH043 on gel</p>
 +
        </td>
 +
        <td class="white arrow" >
 +
            <p>Use for CAR <br/>OE-PCR</p>
 +
        </td>   
 +
        <td class="red arrow" >
 +
            <p>Low yield</p>
 +
        </td>     
 +
  </tr>
 +
    <tr>
 +
        <td class="first rightline"><div class="heightdiv"><h3></span></h3></div></td>
 +
           
 +
        <td class="white arrow" >
 +
            <p>Re-restrict <br/>with high <br/>amount of DNA</p>
 +
        </td>
 +
<td class="white arrow" >
 +
            <p>Extract &<br/> purify <br/>from gel</p>
 +
        </td>
 +
<td class="red arrow" >
 +
            <p>Low yield</p>
 +
        </td>
 +
</tr>
 +
    <tr>
 +
        <td class="first rightline"><div class="heightdiv bottomline"><h3></h3></div></td>
 +
           
 +
        </td>
 +
        <td class="white arrow" >
 +
            <p style="font-size:12px">New o/n culture <br/>of AH043 (needed <br/>more backbone)</p>
 +
        </td>
 +
<td class="blue arrow" >
 +
            <p>MiniPrep</p>
 +
        </td>
 +
<td class="white arrow" >
 +
            <p>Remove insert</p>
 +
        </td>
 +
<td class="white arrow" >
 +
            <p>Use for CAR <br/>NEBuilder</p>
 +
        </td>
 +
</tr>
 +
    <tr>
 +
        <td class="first rightline"><div class="heightdiv bottomline"><h3>Violacein Plasmid<span style="font-size:20px">Construction</span></h3></div></td>
 +
           
 +
        </td>
 +
        <td class="blue arrow" >
 +
            <p>PCR purification<br/>for VioA <br/>2nd PCR</p>
 +
        </td>
 +
<td class="white arrow" >
 +
            <p>Stored in -20°C</p>
 +
        </td>
 +
</tr>
 +
    <tr>
 +
        <td class="first rightline"><div class="heightdiv bottomline"><h3>Cellulose Plasmid <span style="font-size:20px">Construction</span></h3></div></td>
 +
           
 +
        <td class="blue arrow" >
 +
            <p>PCR purification <br/> for VioA <br/> 2nd PCR</p>
 +
        </td>
 +
<td class="white arrow" >
 +
            <p>Stored in -20°C</p>
 +
        </td>
 +
</tr>
 +
    <tr>
 +
        <td class="first rightline"><div class="heightdiv"><h3>Amph protein</h3></div></td>
 +
        <td class="white arrow">   
 +
        <p style="font-size:12px">Gradient PCR <br/> P044&P045, P046&P051, <br/> P045&P046</p>
 +
        </td>
 +
        <td class="white arrow">
 +
        <p>Extract & <br/> purify from gel<br/> P044&P045</p>
 +
        </td>
 +
<td class="white arrow" >
 +
            <p>DNA not clean</p>
 +
        </td>
 +
<td class="white arrow" >
 +
            <p>Store in -20°C</p>
 +
        </td>
 +
</tr>
 +
    <tr>
 +
        <td class="first rightline"><div class="heightdiv bottomline"><h3></h3></div></td>     
 +
        <td>
 +
        </td>
 +
<td class="whitedown arrow" >
 +
            <p style="font-size:12px">Stored P046&P051 <br/>and P045&P046 <br/>product gels <br/>to +4°C</p>
 +
        </td>
 +
</tr>
 +
  <tr>
 +
        <td class="first rightline"><div class="heightdiv"><h3>GFP protein</h3></div></td>
 +
       
 +
        <td class="red arrow" >
 +
            <p style="color:red">Re-do colony <br/>PCR for CAR3 <br/>successful TOPO colony</p>
 +
        </td>
 +
<td class="red arrow" >
 +
            <p style="color:red">Re-do <br/>colony PCR</p>
 +
        </td>
 +
</tr>
 +
    <tr>
 +
        <td class="first rightline"><div class="heightdiv bottomline"><h3>Preparations</h3></div></td>
 +
 +
        <td class="white arrow" >
 +
            <p style="font-size:12px">Restrict and <br/>ligate AH006 <br/>into AH031&AH033</p>
 +
        </td>
 +
<td class="white arrow" >
 +
            <p>Transform into TOP10</p>
 +
        </td>
 +
</tr>
 +
 +
    <tr>
 +
        <td class="first rightline"><div class="heightdiv"><h3>Propane <span style="font-size:20px">Measurement</span></h3></div></td>
 +
 
 +
        <td class="white arrow" >
 +
            <p>o/n culture <br/>of Pauli's <br/>propane strain</p>
 +
        </td>
 +
<td class="white arrow" >
 +
            <p>Propane measurement</p>
 +
        </td>
 +
<td class="white arrow" >
 +
            <p>Results <br/>available <br/>next week</p>
 +
        </td>
 +
</tr>
 +
</table>
 +
 
<h2 id="week13">Week 13</h2>
 
<h2 id="week13">Week 13</h2>
 
<h2 id="week14">Week 14</h2>
 
<h2 id="week14">Week 14</h2>

Revision as of 07:11, 17 August 2015

Lab Book

An example of what the table could look like:

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?

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Here is my text

Here is my
text, there
is lot of it

Ado?

Here is my text

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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

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

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

Re-do colony
PCR for CAR3
successful TOPO colony

Re-do
colony PCR

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