Team:Freiburg/Notebook/29 August

From 2011.igem.org

(Difference between revisions)
(→PCR)
(→PCR to amplify Green light Promotor PcpcG)
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'''Investigators:Julia'''
'''Investigators:Julia'''
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'''PCR'''
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{| style="border-spacing:0;"
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| style="border-top:0.0139in solid #808080;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| Name: Julia
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| style="border:0.0139in solid #808080;padding:0.0194in;"| Date: 29.08.2011
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|-
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| colspan="2"  style="border:0.0139in solid #808080;padding:0.0194in;"| Project Name: Amplification of Green light Promotor
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|}
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PCR-Mixture for one Reaction:
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For a 50 µl reaction use
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{| style="border-spacing:0;"
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| style="border-top:0.0139in solid #808080;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| 32,5µl
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| style="border-top:0.0139in solid #808080;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| H20
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| style="border:0.0139in solid #808080;padding:0.0194in;"|
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|-
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| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| 10µl
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| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| 5x Phusion Buffer
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| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:0.0139in solid #808080;padding:0.0194in;"|
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|-
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| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| 2.5µl
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| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| Primer     fw
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| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:0.0139in solid #808080;padding:0.0194in;"|  tatgaattcgcggccgcttctagaCCATTGTGCTTTTCTCTATCAACC
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|-
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| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| 2.5µl
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| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| Primer     dw
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| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:0.0139in solid #808080;padding:0.0194in;"|  tatctgcagcggccgctactagtaACTTAAAAGTTGTTTAATGTCCAGCC
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|-
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| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| 1µl
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| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| dNTPs
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| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:0.0139in solid #808080;padding:0.0194in;"|
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|-
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| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| 1µl
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| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| DNA-Template
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| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:0.0139in solid #808080;padding:0.0194in;"|  original Synechocystis genome
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|-
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| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| 0.5 µl
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| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:none;padding:0.0194in;"| Phusion
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| style="border-top:none;border-bottom:0.0139in solid #808080;border-left:0.0139in solid #808080;border-right:0.0139in solid #808080;padding:0.0194in;"|  
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|}
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What program do you use?
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To confirm the PCR-Product has the correct size, load 2 µl of the sample onto an agarose-gel.
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===Testdigest of quickchanged CcaR===
===Testdigest of quickchanged CcaR===

Revision as of 12:34, 30 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

PCR to amplify Green light Promotor PcpcG

Investigators:Julia PCR


Name: Julia

 

Date: 29.08.2011
Project Name: Amplification of Green light Promotor

 

PCR-Mixture for one Reaction:

For a 50 µl reaction use


32,5µl H20
10µl 5x Phusion Buffer
2.5µl Primer     fw  tatgaattcgcggccgcttctagaCCATTGTGCTTTTCTCTATCAACC
2.5µl Primer     dw  tatctgcagcggccgctactagtaACTTAAAAGTTGTTTAATGTCCAGCC
1µl dNTPs
1µl DNA-Template  original Synechocystis genome
0.5 µl Phusion  

 

What program do you use?

 

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

 

Testdigest of quickchanged CcaR

Investigators:Julia,with help of the team

blue light receptor

3A-assembly

Investigators: Sandra

Digestion I:

  • Lovtap
  • Not-Gate
  • pSB1A3
  • pSB1T3

stored in the blue light box.

Ligation

  • ♥-A3 Not
  • ♥-A3 nOt
  • ♥-A3 noT
  • ♥-T3 Not
  • ♥-T3 nOt
  • ♥-T3 noT

stored in the ligation box.

Digestion II:

Also digestion with new amplified vector

  • pSB1C3

Lovtap was digested again this time also with DpnI. So for this ligation the ♥-PCR3A, digested with DpnI, and pSB1C3 (new amplified with PCR, already digested with DpnI) and Not, nOt, noT were used.

  • ♥-C3 Not
  • ♥-C3 nOt
  • ♥-C3 noT

Transformation

Investigators: Sophie

transformation of:

  • ♥-A3 Not
  • ♥-A3 nOt
  • ♥-A3 noT
  • ♥-T3 Not
  • ♥-T3 nOt
  • ♥-T3 noT

Incubation over night at 37°C.

PCR

Investigator: Sophie


PCR


Name:


Date:


Continue from Experiment: new


Project Name:

more NOT-gate with mutated restriction site (NHE I instead of Spe I)

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 P 24 (1:10)
2.5µl Primer dw P 79 (1:10)
1µl dNTPs of Template DNA
1µl DNA-Template

M45 (Bba_Q04400)

0.5 µl Phusion (add in the end)

The program we used: RT 2step (see last PCR of NOT-gate)
Freiburg11.08.11.jpg

red light receptor

NAME OF YOUR EXPERIMENT

Investigators:NAME



Lysis cassette

NAME OF YOUR EXPERIMENT

Investigators:NAME


Precipitator

NAME OF YOUR EXPERIMENT

Investigators: NAME

                       

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