Team:Freiburg/Notebook/22 August

From 2011.igem.org

(Difference between revisions)
(→PCR)
(→NAME OF YOUR EXPERIMENT)
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==<span style="color:green;">green light receptor</span>==
==<span style="color:green;">green light receptor</span>==
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===NAME OF YOUR EXPERIMENT===
+
===second quickchange of CcaS===
-
'''Investigators:NAME'''
+
'''Investigators:Julia'''
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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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&nbsp;
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| style="border:0.0139in solid #808080;padding:0.0194in;"| Date: 22.08.11
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|-
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| colspan="2"  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;"| &nbsp;
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|-
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| colspan="2"  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;"| Project Name: second quickchange of CcaS
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|}
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&nbsp;
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 +
PCR-Mixture for one Reaction:
 +
 +
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
 +
| 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;"| &nbsp;
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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
 +
| 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
 +
| 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;"| &nbsp;
 +
 +
|-
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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&nbsp;&nbsp;&nbsp;&nbsp; fw
 +
| 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;"| CGGATCATCAATGGGCTCC (P64)
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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&nbsp;&nbsp;&nbsp;&nbsp; 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;"| GGTCCGCGCATTCTCTATGTCAATGAAGCAT (P65)
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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;"| &nbsp;
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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;"| qS50 and qS51 (5ng/ µl)
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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 (add in the end)
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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;"| &nbsp;
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|}
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&nbsp;
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What program do you use?
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{| style="border-spacing:0;"
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|| Temperature
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|| time (min)
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|| No. of cycles
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||
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|-
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|| <div align="right">95</div>
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|| <div align="right">5:00</div>
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|| <div align="right">1</div>
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||
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|-
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|| <div align="right">95</div>
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|| <div align="right">0:15</div>
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|| &nbsp;
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||
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|-
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|| <div align="right">60</div>
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|| <div align="right">0:15</div>
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|| <div align="right">20</div>
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||
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|-
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|| <div align="right">72</div>
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|| <div align="right">1:15</div>
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|| &nbsp;
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||
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|-
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|| <div align="right">72</div>
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|| <div align="right">5:00</div>
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|| <div align="right">1</div>
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||
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|-
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|| <div align="right">4</div>
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|| <div align="right">60:00:00</div>
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|| <div align="right">1</div>
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||
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|-
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||
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||
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||
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|-
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|| &nbsp;
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|| &nbsp;
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|| &nbsp;
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|| &nbsp;
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|}
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After PCR: Digestion with DpnI in order to get of the old plasmids :
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 +
5 µl NEB buffer 4
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 +
5 µl BSA (10x)
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2 µl DpnI
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38 µl of PCR Product
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Incubation for 1,5h, deactivation at 80°C&nbsp; for 20 min.
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 +
Transformation with 10 µl of the digested PCR product. Cells were plated out on Kanamycin plates( at 23:00 o’clock ).
==<span style="color:blue;">blue light receptor</span>==
==<span style="color:blue;">blue light receptor</span>==

Revision as of 13:01, 24 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

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

Investigators: Rüdiger, Sandra

Transformation of 61,62 and 63.
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.

                       

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