Team:Freiburg/Notebook/16 August
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{{:Team:Freiburg/Templates/header}} | {{:Team:Freiburg/Templates/header}} | ||
+ | <html> | ||
+ | <div id="notebook-page-header"> | ||
+ | <div id="notebook-back" width="100px" > | ||
+ | <a href="https://2011.igem.org/Team:Freiburg/Notebook/15_August">Previous entry</a> | ||
+ | </div> | ||
+ | <div id="notebook-title"> | ||
+ | <a href="https://2011.igem.org/Team:Freiburg/Notebook"> 16 August </a> | ||
+ | </div> | ||
+ | <div id="notebook-next"> | ||
+ | <a href="https://2011.igem.org/Team:Freiburg/Notebook/17_August">Next entry</a> | ||
+ | </div> | ||
+ | </div> | ||
+ | </html> | ||
+ | |||
==<span style="color:green;">green light receptor</span>== | ==<span style="color:green;">green light receptor</span>== | ||
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==<span style="color:red;">red light receptor</span>== | ==<span style="color:red;">red light receptor</span>== | ||
- | === | + | ===continuing with PCR of cph8=== |
- | '''Investigators: | + | '''Investigators:Julia''' |
+ | <br/> | ||
+ | 2µl of the PCR reaction from 15th Aug. were loaded onto a gel.<br/> | ||
+ | But there was no band. Jakob is going to repeat the PCR.<br/> | ||
+ | ===3a-assembly with promotor+ribosome binding site infront of pcyA and ho1=== | ||
+ | '''Investigators:Julia''' | ||
+ | '''Digestion''' | ||
+ | *PR1 in pSB1C3 | ||
+ | *PR2 in pSB1C3 | ||
+ | *pcyA (pSB1T3) | ||
+ | #pcyA+terminator(d)in pSB1T3 | ||
+ | |||
+ | *ho1+Terminator (pSB1T3) | ||
+ | *vector (pSB1K3) | ||
+ | |||
+ | |||
+ | Amount of DNA and H20: | ||
+ | |||
+ | {| cellpadding="10" cellspacing="0" border="1" | ||
+ | |Sample | ||
+ | |500(ng)/DNA concentration (ng/μl) | ||
+ | |H20 μl | ||
+ | |- | ||
+ | |pcyATd | ||
+ | |2.8 | ||
+ | |35.2 | ||
+ | |- | ||
+ | |pcyaTb | ||
+ | |2.9 | ||
+ | |35.1 | ||
+ | |- | ||
+ | |PR2 | ||
+ | |7.2 | ||
+ | |30.8 | ||
+ | |- | ||
+ | |PR1 | ||
+ | |6.1 | ||
+ | |31.9 | ||
+ | |- | ||
+ | |S22a | ||
+ | |2,5 | ||
+ | |35.5 | ||
+ | |- | ||
+ | |pSB1K3 | ||
+ | |20 | ||
+ | |18 | ||
+ | |} | ||
+ | |||
+ | |||
+ | Enzymes necessary for digestion: | ||
+ | |||
+ | {|cellpadding="10" cellspacing="0" border="1" | ||
+ | | | ||
+ | |PR1/PR2 | ||
+ | |pcyA/S22a | ||
+ | |vector | ||
+ | |- | ||
+ | |enzyme 1 | ||
+ | |EcoRI | ||
+ | |XbaI | ||
+ | |EcoRI | ||
+ | |- | ||
+ | |enzyme 2 | ||
+ | |SpeI | ||
+ | |PstI | ||
+ | |PstI | ||
+ | |} | ||
+ | |||
+ | |||
+ | -incubated for 1 hours at 37°C.<br/> | ||
+ | -heated for 20 minutes at 80°C | ||
+ | |||
+ | |||
+ | '''Ligation''' | ||
+ | |||
+ | Ligation of: | ||
+ | |||
+ | {|cellpadding="10" cellspacing="0" border="1" | ||
+ | | | ||
+ | |PcyATd | ||
+ | |||
+ | |S22a (ho1) | ||
+ | |- | ||
+ | |PR1 | ||
+ | |'''1 pTd''' | ||
+ | |'''1 pTb''' | ||
+ | |||
+ | |- | ||
+ | |PR2 | ||
+ | |'''2 pTd''' | ||
+ | |'''2 pTb''' | ||
+ | |||
+ | |} | ||
+ | |||
+ | ===PCR=== | ||
+ | |||
+ | '''Investigator: Jakob''' | ||
+ | |||
+ | |||
+ | '''PCR''' | ||
+ | |||
+ | |||
+ | {| 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: | ||
+ | |||
+ | Jakob | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Date: | ||
+ | |||
+ | 16.08.2011 | ||
+ | |||
+ | |- | ||
+ | | colspan="2" style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Continue from Experiment: 12.08.2011 | ||
+ | |||
+ | Order primers | ||
+ | |||
+ | |- | ||
+ | | colspan="2" style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Project Name: | ||
+ | |||
+ | Red light receptor, amplify Cph8 | ||
+ | |||
+ | |} | ||
+ | PCR-Mixture for one Reaction: | ||
+ | |||
+ | For a 50 µl reaction use | ||
+ | |||
+ | |||
+ | {| style="border-spacing:0;" | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 32,5µl | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| H<sub>2</sub>0 | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Name | ||
+ | |||
+ | |- | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 10µl | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 5x Phusion Buffer | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| of Primer | ||
+ | |||
+ | |- | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 2.5µl | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Primer fw | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| P 58 (1:10) | ||
+ | |||
+ | |- | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 2.5µl | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Primer dw | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| P 59 (1:10) | ||
+ | |||
+ | |- | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 1µl | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| dNTPs | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| of Template DNA | ||
+ | |||
+ | |- | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 1µl | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| DNA-Template | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| JT122 5ng/µl | ||
+ | |||
+ | |- | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 0.5 µl | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Phusion (add in the end) | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| | ||
+ | |||
+ | |} | ||
+ | The program we used: | ||
+ | |||
+ | |||
+ | {| style="border-spacing:0;" | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>Temp.</center> | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>Time</center> | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Nr. of cycles | ||
+ | |||
+ | |- | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>98°</center> | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>5´</center> | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>1x</center> | ||
+ | |||
+ | |- | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>98°</center> | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>30´´</center> | ||
+ | |rowspan="3" style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>10x</center> | ||
+ | |||
+ | |- | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>65°</center> | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>30´´</center> | ||
+ | |||
+ | |- | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>72°</center> | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>1´5´´</center> | ||
+ | |||
+ | |- | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>98°</center> | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>30´´</center> | ||
+ | | rowspan="2" style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>20x</center> | ||
+ | |||
+ | |- | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>72°</center> | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>1´5´´</center> | ||
+ | |||
+ | |- | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>72°</center> | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>7´</center> | ||
+ | | rowspan="2" style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>1x</center> | ||
+ | |||
+ | |- | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>4°</center> | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>∞</center> | ||
+ | |} | ||
+ | <br/> | ||
+ | '''We got the information for the program and the primer sequences from the iGEM Team Uppsala Sweden.''' | ||
==<span style="color:orange;">Lysis cassette</span>== | ==<span style="color:orange;">Lysis cassette</span>== | ||
- | === | + | ===2A Assembly like August 15=== |
- | '''Investigators: | + | '''Investigators:Theo''' |
+ | |||
+ | |||
+ | This time I used all the amount of S15 plasmid DNA we had, did an overnight restriction and run the gel.<br> | ||
+ | [[File:Freiburg11 1608gelextraction.png]] | ||
+ | <br> | ||
+ | extraction as per SOP and ligation with S4 (cut with SpeI, PstI), Transformation in competent cells. | ||
==<span style="color:grey;">Precipitator</span>== | ==<span style="color:grey;">Precipitator</span>== |
Latest revision as of 01:09, 22 September 2011