Team:Freiburg/Notebook/18 August

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{{:Team:Freiburg/Templates/header}}
{{:Team:Freiburg/Templates/header}}
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<div id="notebook-page-header">
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<a href="https://2011.igem.org/Team:Freiburg/Notebook/17_August">Previous entry</a>
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<div id="notebook-title">
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<a href="https://2011.igem.org/Team:Freiburg/Notebook"> 18 August </a>
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</div>
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<div id="notebook-next">
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<a href="https://2011.igem.org/Team:Freiburg/Notebook/19_August">Next entry</a>
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==<span style="color:green;">green light receptor</span>==
==<span style="color:green;">green light receptor</span>==
-
===NAME OF YOUR EXPERIMENT===
+
===Quickchange===
-
 
+
-
'''Investigators:NAME'''
+
-
 
+
 +
'''Investigators:Jakob'''
==<span style="color:blue;">blue light receptor</span>==
==<span style="color:blue;">blue light receptor</span>==
Line 20: Line 32:
==<span style="color:red;">red light receptor</span>==
==<span style="color:red;">red light receptor</span>==
-
===NAME OF YOUR EXPERIMENT===
+
picked colonies from yesterdays trnasformation. <br/>
-
 
+
<br/>
-
'''Investigators:NAME'''
+
new PCR in order to again cph8 from pJT122.<br/>last time PCR did not work.
-
 
+
-
 
+
-
 
+
==<span style="color:orange;">Lysis cassette</span>==
==<span style="color:orange;">Lysis cassette</span>==
-
===NAME OF YOUR EXPERIMENT===
+
===2A assembly===
-
'''Investigators:NAME'''
+
'''Investigators:Theo'''
 +
Sent for sequencing
==<span style="color:grey;">Precipitator</span>==
==<span style="color:grey;">Precipitator</span>==
-
'''Miniprep'''
+
===Miniprep===
zymo Kit
zymo Kit
Line 58: Line 68:
|}
|}
-
Procedure:
 
-
 
-
The following procedures are carried out at a room temperature. All centrifugation steps should be performed between 11,000 – 16,000 x g
 
-
 
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1.Centrifuge 0.5 – 5 ml of bacterial culture in a clear 1.5 ml tube at full speed for 15 – 20 seconds in a microcentrifuge. Discard supernatant.
 
-
 
-
 
-
2.Add 200 µl of '''P1 Buffer '''(Red) to the tube and resuspend pellet completely (i.e., by vortexing or pipetting).
 
-
 
-
 
-
3.Add 200 µl of '''P2 Buffer''' (Green) and mix by inverting the tube 2 – 4 times. Cells are completely lysed when the solution appears clear, purple and viscous. '''Proceed to the next step within 1-2 minutes'''.
 
-
 
-
 
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4.Add 400 µl of '''P3 Buffer''' (Yellow) and mix gently but thoroughly. Do not vortex. The sample will turn yellow when the neutralization is complete. Allow the lysate to incubate at room temperature for 1-2 minutes.
 
-
 
-
 
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5. Centrifuge sample(s) for 2 minutes.
 
-
 
-
6.Place a '''Zymo-Spin IIN''' column in a '''Collection Tube''' and transfer the supernatant from step 5into the '''Zymo-Spin IIN''' column. When pipetting the supernatant be careful not to disturb the green pellet to avoid transferring anz cellular debris to the column.
 
-
 
-
 
-
7.Centrifuge the '''Zymo-Spin IIN/Collection Tube''' assembly for 30 seconds.
 
-
 
-
 
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8.Discard the flow-through in the '''Collection Tube, '''making sure the flow-through does not touch the bottom of the column. Return the '''Zymo-Spin IIN''' column to the '''Collection Tube'''
 
-
 
-
 
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9.Add 200 µl of '''Endo-Wash-Buffer''' to the column and centrifuge for 30 seconds.
 
-
 
-
 
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10. Add 400 µl of '''Plasmid Wash Buffer''' to the column. Centrifuge for 1 minute.
 
-
 
-
11. Transfer the column into a clean 1.5 ml microcentrifuge tube and then add 30 µl of '''DNA Elution Buffer''' to the column. Centrifuge for 30 seconds to elute the plasmid DNA.
 
Line 128: Line 105:
|}
|}
-
Testdigest
+
<br/>
 +
===Testdigest===
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Add 1 μl Loading dye buffer and load the gel.
Add 1 μl Loading dye buffer and load the gel.
-
Take a picture of the gel, print picture and label the lanes!  
+
Take a picture of the gel, print picture and label the lanes! <br/>
 +
[[File:Freigem18.8.11.jpg]]
 +
<br/>
 +
===3A-assembly of IPTG-promoter, pbd-GFP and Cm-vector===
 +
<br/>
 +
 +
Investigators: Sophie
 +
 +
'''Digestion'''
 +
 +
Amount of DNA and H20:
 +
 +
{| cellpadding="10" cellspacing="0" border="1"
 +
|Sample
 +
|DNA μl
 +
|H20 μl
 +
|-
 +
|IPTG-Promoter
 +
|2,5
 +
|33
 +
|-
 +
|GFP-pbd
 +
|3
 +
|36
 +
|-
 +
|pSB1A3
 +
|20
 +
|18
 +
|}
 +
 +
 +
Enzymes necessary for digestion:
 +
 +
{|cellpadding="10" cellspacing="0" border="1"
 +
|
 +
|GFP-pbd: 4 4 4 or 6 6 8
 +
|IPTG-Promoter: S54
 +
|vector: psB1C3
 +
|-
 +
|enzyme 1
 +
|EcoRI
 +
|XbaI
 +
|EcoRI
 +
|-
 +
|enzyme 2
 +
|NheI
 +
|PstI
 +
|PstI
 +
|}
 +
 +
 +
*Incubation at 37°C for 6 hours
 +
*20 minutes at 80°C
 +
<br/>
 +
<br/>
 +
 +
===Ligation===
 +
 +
 +
{| 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: Sophie
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Date: 18.08.11
 +
 +
|-
 +
| 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: 18.08.11 Name: Sophie
 +
 +
Experiment: Cloning
 +
 +
|-
 +
| 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 with Cm-vector
 +
 +
|}
 +
'''Procedure'''
 +
 +
 +
PCR tube:
 +
 +
total volume 20 μl
 +
 +
 +
# add H<sub>2</sub>O (17 μl -X-Y-Z)
 +
# add 2 μl Ligase Buffer 10x
 +
# add Insert 1, Insert 2(when proceeding from 3A digestion use 2 μl of each)
 +
# add Vector (20ng needed. When proceeding from 3A digestion use 2 μl)
 +
# Add 1 μl T4-DNA Ligase
 +
# Incubate 10-30 min at room temperature
 +
# heat for 20 minutes at 80°C
 +
# store at -20°C or directly proceed to transformation
 +
 +
 +
{| style="border-spacing: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;"| Name of part
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Ratio Insert:Vector
 +
 +
<nowiki>= 3:1 or 1:1</nowiki>
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Volume (μl)
 +
 +
|-
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| X insert 1
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| S54
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| both
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 2 or 3
 +
 +
|-
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Y insert 2
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| PR 444 bzw. PR 668
 +
| 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;"| 2 or 3
 +
 +
|-
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| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Z vector
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Psb1A3
 +
| 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;"| 2 or 1
 +
 +
|-
 +
| 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="background-color:#bfbfbf;border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"|
 +
| style="background-color:#bfbfbf;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;"| 11
 +
 +
|}
 +
'''Documentation:'''
 +
 +
Why are you doing this experiment? Where are your parts stored? Name the parts for ligation
 +
 +
 +
{| style="border-spacing:0;"
 +
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Stored in Ligation-box, single parts stored in minipreps, verdaut-box
 +
 +
name of parts see above.
 +
 +
|}
 +
 +
===PCR===
 +
 +
'''Investigators: Rüdiger'''
 +
 +
{|cellpadding="10" cellspacing="0" border="1"
 +
|name
 +
|templates
 +
|primer
 +
|-
 +
|a
 +
|pGex-6-P-1a
 +
|P62+P63
 +
|-
 +
|b
 +
|pGex-6-P-1b
 +
|´´
 +
|-
 +
|a10
 +
|pGex-G-P-1a
 +
|´´
 +
|-
 +
|b10
 +
|pGex-6-P-1b
 +
|´´
 +
|}
 +
PCR did not work. New primer design.
{{:Team:Freiburg/Templates/footer}}
{{:Team:Freiburg/Templates/footer}}

Latest revision as of 01:09, 22 September 2011


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


Contents

green light receptor

Quickchange

Investigators:Jakob

blue light receptor

NAME OF YOUR EXPERIMENT

Investigators:NAME



red light receptor

picked colonies from yesterdays trnasformation.

new PCR in order to again cph8 from pJT122.
last time PCR did not work.

Lysis cassette

2A assembly

Investigators:Theo


Sent for sequencing

Precipitator

Miniprep

zymo Kit


Name:Sophie


Date:18.08.11
Continue from Experiment: Cloning: ε5 was empty and the products from the cloning of 12.08.11 contain GFP-pbd too. (Date): 12.08.11

(Name): Sophie

Project Name: inducible Promoter with pbd with cm-vector


Documentation:

Why are you doing this experiment? Name the parts which you extract.

ε5 was empty and the products from the cloning of 12.08.11 contain GFP-pbd too.

Name of the parts: GFP-pbd 4 4 4, GFP-pbd 4 1 3, GFP-pbd 6 6 8, GFP-pbd 4 2 2


Describe your results and mistakes and measure the DNA concentration with the Nanodrop and note the results.


GFP-pbd 4 4 4: 164,1 ng/µl

GFP-pbd 4 1 3: 100,8 ng/µl

GFP-pbd 6 6 8: 163,5 ng/µl

GFP-pbd 4 2 2: 144,4 ng/µl

How did you label your probes and where are they stored?


Labelled GFP-pbd 4 4 4, GFP-pbd 4 1 3, GFP-pbd 6 6 8, GFP-pbd 4 2 2

stored in -20 until sequencing


Testdigest

Name: Sophie


Date: 18.08.11
Continue from Experiment: Miniprep (Date): 18.08.11

(Name): Sophie

Project Name:inducible promoter for pbd with cm-vector

For one reaction you need: For Mastermix: Number of samples+2extra


4μl H2O 20
1μl Buffer, NEB4 5
1μl BSA (10x) 5
0,5 μl Enzym 1 2,5
0,5 μl Enzym 2 2,5
3 μl DNA

10 μl total volume


Give 3 μl of DNA in an eppi and add 7μl of the mastermix.

Incubate for about 1h at 37°C.


Add 1 μl Loading dye buffer and load the gel.

Take a picture of the gel, print picture and label the lanes!
Freigem18.8.11.jpg

3A-assembly of IPTG-promoter, pbd-GFP and Cm-vector


Investigators: Sophie

Digestion

Amount of DNA and H20:

Sample DNA μl H20 μl
IPTG-Promoter 2,5 33
GFP-pbd 3 36
pSB1A3 20 18


Enzymes necessary for digestion:

GFP-pbd: 4 4 4 or 6 6 8 IPTG-Promoter: S54 vector: psB1C3
enzyme 1 EcoRI XbaI EcoRI
enzyme 2 NheI PstI PstI


  • Incubation at 37°C for 6 hours
  • 20 minutes at 80°C



Ligation

Name: Sophie Date: 18.08.11
Continue from Date: 18.08.11 Name: Sophie

Experiment: Cloning

Project Name: inducible Promoter for pbd with Cm-vector

Procedure


PCR tube:

total volume 20 μl


  1. add H2O (17 μl -X-Y-Z)
  2. add 2 μl Ligase Buffer 10x
  3. add Insert 1, Insert 2(when proceeding from 3A digestion use 2 μl of each)
  4. add Vector (20ng needed. When proceeding from 3A digestion use 2 μl)
  5. Add 1 μl T4-DNA Ligase
  6. Incubate 10-30 min at room temperature
  7. heat for 20 minutes at 80°C
  8. store at -20°C or directly proceed to transformation


Name of part Ratio Insert:Vector

= 3:1 or 1:1

Volume (μl)
X insert 1 S54 both 2 or 3
Y insert 2 PR 444 bzw. PR 668 2 or 3
Z vector Psb1A3 2 or 1
H2O 11

Documentation:

Why are you doing this experiment? Where are your parts stored? Name the parts for ligation


Stored in Ligation-box, single parts stored in minipreps, verdaut-box

name of parts see above.

PCR

Investigators: Rüdiger

name templates primer
a pGex-6-P-1a P62+P63
b pGex-6-P-1b ´´
a10 pGex-G-P-1a ´´
b10 pGex-6-P-1b ´´

PCR did not work. New primer design.

                       

Retrieved from "http://2011.igem.org/Team:Freiburg/Notebook/18_August"