Team:Freiburg/Notebook/30 August

From 2011.igem.org

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(→NAME OF YOUR EXPERIMENT)
(→Cloning)
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==<span style="color:grey;">Precipitator</span>==
==<span style="color:grey;">Precipitator</span>==
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===Cloning===
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'''Digestion'''
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'''Investigators: Sophie'''
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<br/> Project name: inducible promoter for pbd <br/>
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{| style="border-spacing:0;"
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again cloning of IPTG-inducible Promoter, new psb1C3-PCR product and ε5 (GFP-pbd) but this time I use Antarctic Phosphatase in the last hour of the digestion of the vector to avoid re-ligation. For 50μL reaction volume use 5,6μL Antarctic Phosphatase buffer and 1μL Antarctic Phosphatase.
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Name: Sophie
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{{:Team:Freiburg/Templates/footer}}
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Date: 31.08.11
 +
 
 +
|-
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| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Continue from Date: 30.08.11 Name: Sophie
 +
 
 +
Experiment: new
 +
 
 +
|-
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Project Name:inducible promoter for pbd (iGEM standard)
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 +
|}
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Procedure
 +
 
 +
 
 +
# add H<sub>2</sub>O (38μl-DNA )
 +
# 5 μl NEB4 buffer  (stored at iGEM’s, -20°C)
 +
# 5 μl 10x BSA  (used 1:10 diluted sample stored at iGEM’s, -20°C)
 +
# DNA (500 ng) (Vector:Insert ratio 1:3 in following Ligation)
 +
# 1 μl restriction enzymes  (stored at iGEM’s, -20°C)
 +
# heat for 1-2 hours 37°C (6 hours if time)
 +
# heat for 20 minutes 80°C (inactivation of enzymes)
 +
# keep at 4°C if you cannot continue
 +
 
 +
Measured DNA-concentration with Nanodrop to calculate the volume of DNA to do the digestion:
 +
 
 +
 
 +
{| style="border-spacing:0;"
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Sample Name
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| DNA concentration (μg/μl)
 +
 
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| GFP-pbd
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 164
 +
 
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| IPTG-promoter ε 5
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 217
 +
 
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| psB1C3
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 58
 +
 
 +
|-
 +
| 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;"|
 +
 
 +
|-
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| 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;"|
 +
 
 +
|-
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
 
 +
|-
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
 
 +
|}
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Restriction enzymes you need to cut the vector, insert1 and insert 2:
 +
 
 +
 
 +
{| style="border-spacing:0;"
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| '''Components'''
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| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>'''Vector (μl)'''</center>
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>'''Insert1 and 2 (μl)'''</center>
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 +
|-
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| DNA (500ng)
 +
| colspan="2"  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;"|
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| colspan="0"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
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 +
|-
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| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| BSA (10x) (5μl)
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| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
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| colspan="0"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
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 +
|-
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| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| NEB4 Buffer (5μl)
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
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| colspan="0"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
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 +
|-
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| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Enzyme 1 (1μl)
 +
| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Eco
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| style="background-color:transparent;border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Eco
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| colspan="0"  style="background-color:transparent;border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Xba
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 +
|-
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| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Enzyme 2 (1μl)
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| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Pst
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| style="background-color:transparent;border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Spe
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| colspan="0"  style="background-color:transparent;border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Pst
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 +
|-
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| colspan="2"  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 (38 μl- DNA)
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| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
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| colspan="0"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
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 +
|-
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| colspan="2"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| In total 50 μl
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| colspan="2"  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;"|
 +
| colspan="0"  style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
 
 +
|}
 +
'''Documentation:'''
 +
 
 +
Why are you doing this experiment? Where are the samples stored? Antibiotica resistance, vector used etc.
 +
 
 +
 
 +
{| style="border-spacing:0;"
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| I want to be able to send the plastic binding domain to the registry and I want it to be expressible.
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Stored in: “Minipreps, verdaut”-box
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Resistance: Cm
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|}

Revision as of 15:22, 31 August 2011


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

Contents

green light receptor

Ligation of Promotor PcpcG in PSB1C3

Investigators:Julia

1. Digestion of PCR Product from yesterday[1]

38µl PCR product
5µl BSA (10x)
5µl NEB buffer 4
1µl EcoRI
1µlPstI

Incubation for 3 hours, afterwards the sample was cleaned with a PCR purification kit from Qiagen.

2.Ligation

11µl H2O
4µl PcpcG
2µl pSB1C3
2µl T4 Ligase buffer
1µl T4 Ligase

Incubation for 3 hours at room temperature.
heat inactivation at 80 degrees for 20 min.

3. transformation

blue light receptor

Transformation

...with ♥-A3-Not, ♥-A3-nOt and ♥-A3-noT </br>

Investigators: Sophie

red light receptor

gel extraction

Investigators:Julia

Lysis cassette

NAME OF YOUR EXPERIMENT

Investigators:NAME


Precipitator

Digestion


Name: Sophie Date: 31.08.11
Continue from Date: 30.08.11 Name: Sophie

Experiment: new

Project Name:inducible promoter for pbd (iGEM standard)

Procedure


  1. add H2O (38μl-DNA )
  2. 5 μl NEB4 buffer (stored at iGEM’s, -20°C)
  3. 5 μl 10x BSA (used 1:10 diluted sample stored at iGEM’s, -20°C)
  4. DNA (500 ng) (Vector:Insert ratio 1:3 in following Ligation)
  5. 1 μl restriction enzymes (stored at iGEM’s, -20°C)
  6. heat for 1-2 hours 37°C (6 hours if time)
  7. heat for 20 minutes 80°C (inactivation of enzymes)
  8. keep at 4°C if you cannot continue

Measured DNA-concentration with Nanodrop to calculate the volume of DNA to do the digestion:


Sample Name DNA concentration (μg/μl)
GFP-pbd 164
IPTG-promoter ε 5 217
psB1C3 58

Restriction enzymes you need to cut the vector, insert1 and insert 2:


Components
Vector (μl)
Insert1 and 2 (μl)
DNA (500ng)
BSA (10x) (5μl)
NEB4 Buffer (5μl)
Enzyme 1 (1μl) Eco Eco Xba
Enzyme 2 (1μl) Pst Spe Pst
H2O (38 μl- DNA)
In total 50 μl

Documentation:

Why are you doing this experiment? Where are the samples stored? Antibiotica resistance, vector used etc.


I want to be able to send the plastic binding domain to the registry and I want it to be expressible.

Stored in: “Minipreps, verdaut”-box

Resistance: Cm

Retrieved from "http://2011.igem.org/Team:Freiburg/Notebook/30_August"