Team:Freiburg/Notebook/22 August

From 2011.igem.org

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==<span style="color:grey;">Precipitator</span>==
==<span style="color:grey;">Precipitator</span>==
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===Transformation===
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{| 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;"| Name:Rüdiger
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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:22.08
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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;"| Continue from Date 19.08. Name Rüdiger
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Experiment Ligation
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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;"| Project Name: Precipitator
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|}
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Procedure
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# take cells from -80°C freezer and put them on ice! (every eppi contains about 400 μl cells)
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# thaw cells on ice 20 minutes
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# pipette 50 μl cells and 2 μl DNA into eppi still on ice!
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# Incubate for 30 minutes on ice
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# Heat at 42°C for 60 sec
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# Incubate on ice for 5 minutes
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# Add 200 μl LB Broth
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# Incubate for 2 hours at 37°C (cells with lysis cassette at 30°C!!)
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# Plate 50 μl and 200μl on two different LB/Agar plates with appropriate antibiotic resistance
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'''Documentation:'''
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Why are you doing this experiment? Name of the sample? Where are they stored? Name the vector with inserts, antibiotika resistance etc.
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{| 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;"| pSB1C3
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61a,b
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62a.,b
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64a,b
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repetition from 20.08.
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Miniprep: Julia 24.05.
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|}
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===Transformation===
===Transformation===

Revision as of 21:27, 21 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

second quickchange of CcaS

Investigators:Julia

Name: Julia

 

Date: 22.08.11
 
Project Name: second quickchange of CcaS

 

PCR-Mixture for one Reaction:

For a 50 µl reaction use


32,5µl H20  
10µl 5x Phusion Buffer  
2.5µl Primer     fw CGGATCATCAATGGGCTCC (P64)
2.5µl Primer     dw GGTCCGCGCATTCTCTATGTCAATGAAGCAT (P65)
1µl dNTPs  
1µl DNA-Template qS50 and qS51 (5ng/ µl)
0.5 µl Phusion (add in the end)  

 

What program do you use?


Temperature time (min) No. of cycles
95
5:00
1
95
0:15
  
60
0:15
20
72
1:15
  
72
5:00
1
4
60:00:00
1
       

After PCR: Digestion with DpnI in order to get of the old plasmids :

5 µl NEB buffer 4

5 µl BSA (10x)

2 µl DpnI

38 µl of PCR Product

Incubation for 1,5h, deactivation at 80°C  for 20 min.

Transformation with 10 µl of the digested PCR product. Cells were plated out on Kanamycin plates( at 23:00 o’clock ).

blue light receptor

Sequencing

Investigators: Sandra

The sequencing showed that the 3A-assembly did not work. We will send the LovTap PCR product to get sequenced. May be the PCR did not work.

PCR

Investigators: Sandra

PCR of Not-Gate.

P 74:

  • actgcagcggccgctgctagca

P 24:

  • tatgaattcgcggccgcttctag


PCR product was loaded onto a gel. Expected lane: 900bp
PCR worked and we got Not-Gate with NEHI and PstI.

Cloning

Investigators: Sophie

new 3A assembly with Sandra's NOT-gate-PCR-product, our LOV-Tap-pcr-product (both mutated with NEH1 restriction sites) and an amp-vector
The digested samples were loaded to a gel. They showed clear bands in the right size. Only the band of the vector was not intense enough, so the vector has to be digested again.

red light receptor

NAME OF YOUR EXPERIMENT

Investigators:NAME



Lysis cassette

NAME OF YOUR EXPERIMENT

Investigators:NAME


Precipitator

Transformation

Name:Rüdiger Date:22.08
Continue from Date 19.08. Name Rüdiger

Experiment Ligation

Project Name: Precipitator

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.


pSB1C3

61a,b

62a.,b

64a,b


repetition from 20.08.


Miniprep: Julia 24.05.


Transformation

Investigators: Rüdiger, Sandra

Transformation of:
61 a,b
62 a,b
63 a,b.

Vector:pSB1C3

Incubation over night.

Cloning for GFP-pbd with IPTG-inucible promoter

Investigator: Sophie
as cloning from 18.08.11 did not work I try it again with longer digestion times.

PCR

Name:Rüdiger


Date: 22.08
Continue from Experiment (Date)-

(Name)

Project Name: Precipitator

PCR-Mixture for one Reaction:

For a 50 µl reaction use


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

What program do you use?

Anneal Gradient from 61-72°c, 15sec.


To confirm the PCR-Product has the correct size, load 2 µl of the sample onto an agarose-gel.


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


Name Template Primer Temperature
1 Precipitator 2 p44+p73 85/69>72
2 Precipitator 4_2 p45+p71 89/68>72
3 Precipitator 4 p46+p72 72
4 pgEX-6-p-1a 1:10 p67+p69 58>61
5 pgEX-6-p-1a 1:10 p67+p70 58>61
6 pgEX-6-p-1a 1:10 p68+p69 62>65
7 pgEX-6-p-1a 1:10 p69+p70 58>61
C Lambda DNA Primers 1,4kb


Investiagators: Rüdiger

                       

Retrieved from "http://2011.igem.org/Team:Freiburg/Notebook/22_August"