Team:Freiburg/Notebook/15 July

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<a href="https://2011.igem.org/Team:Freiburg/Notebook/14_July">Previous entry</a>
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<a href="https://2011.igem.org/Team:Freiburg/Notebook"> 15 July </a>
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==Meeting==
==Meeting==
attendants: Tobi, Theo, Julia, Rüdiger, Jakob, Sandra
attendants: Tobi, Theo, Julia, Rüdiger, Jakob, Sandra
-
===<span style="color:green;">green light receptor</span>===
+
===<span style="color:blue;">blue light receptor</span>===
-
already done:
+
 
 +
already done:  
 +
 
 +
# Transformation of Lov-Tap in cells.
Line 11: Line 29:
To-do:
To-do:
 +
# Cloning of promotor (medium) in Lov-Tap part and perform a transformation. Plating out of the cells on trypthophan-free medium.
 +
<br/>
-
===<span style="color:blue;">blue light receptor</span>===
+
===Quick change===
 +
'''Investigators: Theo'''
already done:
already done:
-
 
+
#repressor part (BBa_K098995) has no bases between RBS (B0034) and start codon (K098997), resulting in no translation.
 +
#*quickchange of the repressor part to insert 6bp between RBS and start codon
To-do:
To-do:
-
===<span style="color:red;">red light receptor</span>===
+
#DpnI digestion to digest the DNA template (methylated DNA) of the PCR to have only the new synthesized DNA strand.
 +
#After digestion with DpnI, transformation of cells with the corrected part (BBa_K08995)
 +
<br/>
-
already done:
+
==<span style="color:orange;">Lysis cassette</span>==
 +
===Digestion of Quickchange===
-
To-do:
+
{| style="border-spacing:0;"
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Name: Theo
-
===<span style="color:orange;">Lysis cassette</span>===
 
-
already done:
 
-
#repressor part (BBa_K098995) has no bases between RBS (B0034) and start codon (K098997), resulting in no translation.
+
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Date: 15.07.2011
-
#*quickchange of the repressor part to insert 6bp between RBS and start codon
+
-
To-do:
+
|-
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Continue from Experiment 14.07.2011
 +
 
 +
Quickchange PCR
 +
 
 +
|-
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Project Name: Correct number of nucleotides between RBS and ATG of temp. sensitive repressor from Lysis Device
 +
 
 +
|}
 +
 
 +
{| style="border-spacing:0;"
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 4,5μl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| H<sub>2</sub>O
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
 
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 4μl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Buffer, NEB4
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
 
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 4μl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| BSA (10x)
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
 
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 1,5 μl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Enzym 1
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| DpnI
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
 
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 21 μl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| DNA
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Quickchange PCR
 +
 
 +
|}
 +
35 μl total volume
 +
 
 +
 
 +
Incubate for about 1h at 37°C + Heat inactivation at 80°C for 20min.
-
#DpnI digestion to digest the DNA template (methylated DNA) of the PCR to have only the new synthesized DNA strand.
 
-
#*After digestion with DpnI, transformation of cells with the corrected part (BBa_K08995)
 
<br/>
<br/>
-
===<span style="color:grey;">Precipitator</span>===
+
===Transformation''' '''===
-
already done:
 
 +
{| style="border-spacing:0;"
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Name: Theo
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Date: 15.07.2011
 +
|-
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Continue from 15.07.2011 Digestion of Quickchange
-
To-do:
+
Experiment
 +
 
 +
|-
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Project Name: Correct number of nucleotides between RBS and ATG of temp. sensitive repressor from Lysis Device
 +
 
 +
|}
 +
Procedure
 +
 
 +
 
 +
# take cells from -80°C freezer and put them on ice! (every eppi contains about 400 μl cells)
 +
# thaw cells on ice 20 minutes
 +
# pipette 50 μl cells and 2 μl DNA into eppi still on ice!
 +
# Incubate for 30 minutes on ice
 +
# Heat at 42°C for 60 sec
 +
# Incubate on ice for 5 minutes
 +
# Add 200 μl LB Broth
 +
# Incubate for 2 hours at 37°C (cells with lysis cassette at 30°C!!)
 +
# Plate 50 μl and 200μl on two different LB/Agar plates with appropriate antibiotic resistance
 +
 
 +
'''Documentation:'''
 +
 
 +
Why are you doing this experiment? Name of the sample? Where are they stored? Name the vector with inserts, antibiotika resistance etc.
 +
 
 +
 
 +
{| style="border-spacing:0;"
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| It was done in ordert to correct the number of nucleotides between RBS and ATG of the temperature sensitive repressor from our Lysis Device, ie to insert 6bp.
 +
 
 +
 
 +
It didn’t work because the false DNA Template was taken (S15 - K124014 - instead of S11 - K098995 -).
 +
 
 +
 
 +
This will be corrected by doing another Quickchange using S11 (ie K098995) as template.
 +
 
 +
|}
-
<br/>
 
<br/>
<br/>
-
==<span style="color:green;">green light receptor</span>==
+
==<span style="color:grey;">Precipitator</span>==
-
===NAME OF YOUR EXPERIMENT===
+
'''PCR'''
-
'''Investigators:NAME'''
 
-
==<span style="color:blue;">blue light receptor</span>==
+
{| style="border-spacing:0;"
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Name:
-
===NAME OF YOUR EXPERIMENT===
+
Ruediger
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Date:
-
'''Investigators:NAME'''
+
18.07.2011
 +
|-
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Continue from Experiment (Date)PCR 1507
 +
(Name) Ruediger
 +
|-
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Project Name:
-
==<span style="color:red;">red light receptor</span>==
+
GFP Pbd
-
===NAME OF YOUR EXPERIMENT===
+
|}
 +
PCR-Mixture for one Reaction:
-
'''Investigators:NAME'''
+
For a 50 µl reaction use
 +
{| style="border-spacing:0;"
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 32,5µl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| H<sub>2</sub>0
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 10µl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 5x Phusion Buffer
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
-
==<span style="color:orange;">Lysis cassette</span>==
+
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 2.5µl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Primer fw
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
-
===NAME OF YOUR EXPERIMENT===
+
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 2.5µl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Primer dw
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
-
'''Investigators:NAME'''
+
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 1µl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| dNTPs
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 1µl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| DNA-Template
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 0.5 µl
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Phusion (add in the end)
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
-
==<span style="color:grey;">Precipitator</span>==
+
|}
 +
What program do you use?
 +
 
 +
10x (95C-41/52C-72C) + 25x ((95C-60C-72C)
 +
 
 +
 
 +
How did you label the PCR-Product, where is it stored and what do you do next?
 +
 
 +
 
 +
Reactions:
 +
 
 +
lane 1
 +
 
 +
quick load braod range marker
 +
 
 +
lane 2
 +
 
 +
empty
 +
 
 +
lane 3
 +
 
 +
P28+P18+M14.1
 +
 
 +
lane 4
 +
 
 +
P28+P19+M14.1
 +
 
 +
lane 5
 +
 
 +
P28+P20+M14.1
 +
 
 +
 
 +
Results:
 +
 
 +
did not work well.
 +
 
 +
Strange band size in lane 3. 4 and 5 did not work.
-
===NAME OF YOUR EXPERIMENT===
+
[[File:Freiburg11_7_16_2011_2_52_42_PM.Jpg]]
 +
[[File:Freiburg11 Phusion ladder.JPG]]
-
'''Investigators: NAME'''
+
{{:Team:Freiburg/Templates/footer}}

Latest revision as of 00:56, 22 September 2011


This is the wiki page
of the Freiburger student
team competing for iGEM 2011.
Thank you for your interest!

Contents

Meeting

attendants: Tobi, Theo, Julia, Rüdiger, Jakob, Sandra

blue light receptor

already done:

  1. Transformation of Lov-Tap in cells.


To-do:

  1. Cloning of promotor (medium) in Lov-Tap part and perform a transformation. Plating out of the cells on trypthophan-free medium.


Quick change

Investigators: Theo

already done:

  1. repressor part (BBa_K098995) has no bases between RBS (B0034) and start codon (K098997), resulting in no translation.
    • quickchange of the repressor part to insert 6bp between RBS and start codon

To-do:

  1. DpnI digestion to digest the DNA template (methylated DNA) of the PCR to have only the new synthesized DNA strand.
  2. After digestion with DpnI, transformation of cells with the corrected part (BBa_K08995)


Lysis cassette

Digestion of Quickchange

Name: Theo


Date: 15.07.2011
Continue from Experiment 14.07.2011

Quickchange PCR

Project Name: Correct number of nucleotides between RBS and ATG of temp. sensitive repressor from Lysis Device
4,5μl H2O
4μl Buffer, NEB4
4μl BSA (10x)
1,5 μl Enzym 1 DpnI
21 μl DNA Quickchange PCR

35 μl total volume


Incubate for about 1h at 37°C + Heat inactivation at 80°C for 20min.


Transformation

Name: Theo Date: 15.07.2011
Continue from 15.07.2011 Digestion of Quickchange

Experiment

Project Name: Correct number of nucleotides between RBS and ATG of temp. sensitive repressor from Lysis Device

Procedure


  1. take cells from -80°C freezer and put them on ice! (every eppi contains about 400 μl cells)
  2. thaw cells on ice 20 minutes
  3. pipette 50 μl cells and 2 μl DNA into eppi still on ice!
  4. Incubate for 30 minutes on ice
  5. Heat at 42°C for 60 sec
  6. Incubate on ice for 5 minutes
  7. Add 200 μl LB Broth
  8. Incubate for 2 hours at 37°C (cells with lysis cassette at 30°C!!)
  9. Plate 50 μl and 200μl on two different LB/Agar plates with appropriate antibiotic resistance

Documentation:

Why are you doing this experiment? Name of the sample? Where are they stored? Name the vector with inserts, antibiotika resistance etc.


It was done in ordert to correct the number of nucleotides between RBS and ATG of the temperature sensitive repressor from our Lysis Device, ie to insert 6bp.


It didn’t work because the false DNA Template was taken (S15 - K124014 - instead of S11 - K098995 -).


This will be corrected by doing another Quickchange using S11 (ie K098995) as template.


Precipitator

PCR


Name:

Ruediger

Date:

18.07.2011

Continue from Experiment (Date)PCR 1507

(Name) Ruediger

Project Name:

GFP Pbd

PCR-Mixture for one Reaction:

For a 50 µl reaction use


32,5µl H20
10µl 5x Phusion Buffer
2.5µl Primer fw
2.5µl Primer dw
1µl dNTPs
1µl DNA-Template
0.5 µl Phusion (add in the end)

What program do you use?

10x (95C-41/52C-72C) + 25x ((95C-60C-72C)


How did you label the PCR-Product, where is it stored and what do you do next?


Reactions:

lane 1

quick load braod range marker

lane 2

empty

lane 3

P28+P18+M14.1

lane 4

P28+P19+M14.1

lane 5

P28+P20+M14.1


Results:

did not work well.

Strange band size in lane 3. 4 and 5 did not work.

Freiburg11 7 16 2011 2 52 42 PM.Jpg Freiburg11 Phusion ladder.JPG

                       

Retrieved from "http://2011.igem.org/Team:Freiburg/Notebook/15_July"