Team:Freiburg/Notebook/29 July
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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/28_July">Previous entry</a> | ||
+ | </div> | ||
+ | <div id="notebook-title"> | ||
+ | <a href="https://2011.igem.org/Team:Freiburg/Notebook"> 29 July </a> | ||
+ | </div> | ||
+ | <div id="notebook-next"> | ||
+ | <a href="https://2011.igem.org/Team:Freiburg/Notebook/1_August">Next entry</a> | ||
+ | </div> | ||
+ | </div> | ||
+ | </html> | ||
==Meeting== | ==Meeting== | ||
- | attendants: | + | attendants: Julia, Manuel, Rüdiger, Sandra, Theo, Tobi |
+ | Time: 9:00 - 11:00 | ||
===<span style="color:green;">green light receptor</span>=== | ===<span style="color:green;">green light receptor</span>=== | ||
already done: | already done: | ||
- | + | *Ccas has to sites where we need to do a mutagenesis (quick change), both were done seperately, but the no colonies showed up (wrong PCR program) | |
+ | *CcaR has one site where we need to do a mutagenesis (quick change), this has been done, colonies were growing | ||
To-do: | To-do: | ||
- | + | *miniprep, digestion and sequencing of the CcaR clones | |
+ | *do the Ccas PCR again | ||
+ | *next steps would be the big assembly wiht pcyA | ||
===<span style="color:blue;">blue light receptor</span>=== | ===<span style="color:blue;">blue light receptor</span>=== | ||
- | already done: PCR of Lov-tap and Not-gate. Not-gate sequencing was good. PCR of Lov-tap did not work | + | already done: |
+ | *PCR of Lov-tap and Not-gate. Not-gate sequencing was good. PCR of Lov-tap did not work the first time and the second time with the temperature gradient PCR | ||
- | To-do: new | + | To-do: |
+ | *creating new primer for the Lov-tap as the old ones had a high probabylity for secondary structures | ||
===<span style="color:red;">red light receptor</span>=== | ===<span style="color:red;">red light receptor</span>=== | ||
already done: | already done: | ||
- | + | *waiting for cph8 from Mexico | |
+ | *for pcyA we did a new transformation from the original plasmid | ||
To-do: | To-do: | ||
+ | *over night culture, miniprep and stock from the pcyA plates | ||
+ | *3-A-Assembly of pcyA and Terminator | ||
===<span style="color:orange;">Lysis cassette</span>=== | ===<span style="color:orange;">Lysis cassette</span>=== | ||
already done: | already done: | ||
- | + | *the quick change didn't work the first time (wrong template DNA) | |
+ | *second time went well, but the transformation was difficult (only 10 colonies) | ||
To-do: | To-do: | ||
- | + | *do the miniprep and the sequencing | |
===<span style="color:grey;">Precipitator</span>=== | ===<span style="color:grey;">Precipitator</span>=== | ||
already done: | already done: | ||
- | + | *GFP-PDB: cloning doesn't work only a few colonies | |
To-do: | To-do: | ||
+ | *ask again if the gene synthesis is done: Rüdiger | ||
+ | *Gibson cloning with the GST-Tag, TEV-site and Promotor-RBS | ||
+ | |||
+ | |||
+ | ===other stuff=== | ||
+ | *deadline for the parts: 10th of september | ||
+ | *name all the files you upload on the wiki with the prefix Freiburg2011_ | ||
+ | *start documenting the lab stuff in the wiki | ||
+ | *still missing: logo and text from hiss, MPI, Serva, Invitrogen | ||
+ | *Modelling: Rüdiger will ask Simon if he can help us along | ||
+ | *sponsoring give-aways: key tag, postcards, movie, pencils | ||
+ | *Lab tracks: Tobi wants to try it | ||
+ | *cooperation with Upsala: Yeahhh! | ||
+ | |||
<br/> | <br/> | ||
+ | |||
+ | |||
+ | ==<span style="color:blue;">blue light receptor</span>== | ||
+ | |||
+ | ===Glycerol stocks of Not-Gate=== | ||
+ | |||
+ | '''Investigators: Sandra''' | ||
+ | |||
+ | M45d was sequenced and we did glycerol stocks of bacteria containing M45d. | ||
+ | |||
+ | <br> | ||
<br/> | <br/> | ||
- | ==<span style="color: | + | ==<span style="color:orange;">Lysis cassette</span>== |
- | === | + | ===Sequencing of Lysis Cassette V.2 (Quickchange-modified K098995 + Lysis genes K124017)=== |
- | + | <br> | |
+ | Test Sequencing showed that the parts were assembled correctly. | ||
+ | <br> | ||
- | + | File #1: You can see the EcoRI, XbaI sites and also the Quickchange-modified K098995 as well as the XbaI-SpeI SCAR and the start of the Lysis genes K124017 | |
- | + | <br> | |
- | + | File #2: You can see part of the Lysis genes K124017 and the SpeI and PstI sites | |
+ | <br> | ||
+ | [https://static.igem.org/mediawiki/2011/c/c7/Freiburg11_seq_qcLysCasV2_1-P8.gb Right-click to download the annotated ape (.gb) file #1] | ||
+ | <br> | ||
- | + | [https://static.igem.org/mediawiki/2011/8/88/Freiburg11_seq_qcLysCasV2_1-P9.gb Right-click to download the annotated ape (.gb) file #2] | |
- | + | <br> | |
- | + | What can immediately be noticed is the absence of a RBS (ribosome binding site) between the lamba cI regulated Promotor (R0051 of K098995) and the holin ATG of K124017. | |
+ | This had skipped our attention at the planing phase and was a source of headaches during the next days... :) | ||
+ | <br> | ||
+ | <br/> | ||
+ | ==Precipitator== | ||
+ | Testdigest | ||
- | |||
- | === | + | {| 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: | ||
+ | |||
+ | Ruediger | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Date: 29.07 | ||
+ | |||
+ | |- | ||
+ | | 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 (Date) Miniprep 28.07 | ||
+ | |||
+ | (Name) | ||
+ | |||
+ | |- | ||
+ | | colspan="2" style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Project Name: GFP Pbd | ||
+ | |||
+ | |} | ||
+ | For one reaction you need: For Mastermix: 12 Number of samples+2extra | ||
+ | |||
+ | |||
+ | {| style="border-spacing:0;" | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 4μl | ||
+ | | 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="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 84 | ||
+ | | 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;"| 1μl | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Buffer, NEB4 | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 14 | ||
+ | | 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;"| 1μl | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| BSA (10x) | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 14 | ||
+ | | 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;"| 0,5 μl | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Enzym 1 | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 7 | ||
+ | | 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;"| 0,5 μl | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| Enzym 2 | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 7 | ||
+ | | 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;"| 3 μl | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| DNA | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| 1 | ||
+ | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| | ||
+ | |||
+ | |} | ||
+ | 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! | |
- | + | nothing worked, no GFP inserts seens anywhere. Gel picture was not saved by machine. | |
+ | -Sequencing: no GFP in constructs - retry by Theo. |
Latest revision as of 01:03, 22 September 2011