"
Team:Freiburg/Notebook/3 August
From 2011.igem.org
(Difference between revisions)
(→Quickchange of BBa_K124017 to insert RBS (B0034) and 6bp in front of the first ATG) |
|||
| (6 intermediate revisions not shown) | |||
| Line 1: | Line 1: | ||
{{: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/2_August">Previous entry</a> | ||
| + | </div> | ||
| + | <div id="notebook-title"> | ||
| + | <a href="https://2011.igem.org/Team:Freiburg/Notebook"> 3 August </a> | ||
| + | </div> | ||
| + | <div id="notebook-next"> | ||
| + | <a href="https://2011.igem.org/Team:Freiburg/Notebook/4_August">Next entry</a> | ||
| + | </div> | ||
| + | </div> | ||
| + | </html> | ||
| + | |||
==<span style="color:green;">green light receptor</span>== | ==<span style="color:green;">green light receptor</span>== | ||
| Line 114: | Line 128: | ||
Add 1 μl Loading dye buffer and load the gel. | Add 1 μl Loading dye buffer and load the gel. | ||
| - | + | * Picture of the gel | |
| Line 128: | Line 142: | ||
<br/> | <br/> | ||
'''Investigator:Jakob''' | '''Investigator:Jakob''' | ||
| - | |||
| - | |||
| Line 219: | Line 231: | ||
*to-do: Miniprep | *to-do: Miniprep | ||
| + | |||
| + | <br> | ||
==<span style="color:orange;">Lysis cassette</span>== | ==<span style="color:orange;">Lysis cassette</span>== | ||
| Line 297: | Line 311: | ||
|- | |- | ||
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>95°C - 5 min</center> | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>95°C - 5 min</center> | ||
| - | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>55°C | + | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>55°C - 15 sec</center> |
| - | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>72°C -5 min</center> | + | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>72°C - 5 min</center> |
| style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>4°C - ∞</center> | | style="border:0.0069in solid #00000a;padding-top:0in;padding-bottom:0in;padding-left:0.075in;padding-right:0.075in;"| <center>4°C - ∞</center> | ||
| Line 317: | Line 331: | ||
<br> | <br> | ||
<br/> | <br/> | ||
| - | |||
| - | ===Digestion of Quickchange=== | + | ===Digestion of Quickchange with DpnI=== |
| Line 368: | Line 381: | ||
Incubate for about 1,5h at 37°C + Heat inactivation at 80°C for 20min. | Incubate for about 1,5h at 37°C + Heat inactivation at 80°C for 20min. | ||
| + | |||
| + | <br/> | ||
| + | ==<span style="color:grey;">Precipitator</span>== | ||
| + | |||
| + | '''Investigators: Theo''' | ||
| + | |||
| + | new 3A-assembly with Kan vector to see if pSB1T3 played a negative role in the last 3A-assembly. | ||
| + | |||
| + | Incubation over night at 16°C. | ||
| + | |||
| + | {{:Team:Freiburg/Templates/footer}} | ||
==<span style="color:grey;">Precipitator</span>== | ==<span style="color:grey;">Precipitator</span>== | ||
Latest revision as of 01:04, 22 September 2011
Contact 
This is the wiki page of the Freiburger student team competing for iGEM 2011. Thank you for your interest!
Contents |
green light receptor
Testdigest
Investigators:Jakob, Julia
| Name:
Jakob | Date:
02.08.2011 |
| Continue from Experiment 02.08.2011
Miniprep | |
| Project Name: green light receptor, testdigest of CcaR with BamHI | |
For one reaction you need: For Mastermix: Number of samples+2extra
| 4μl | H2O | 20 | |
| 1μl | Buffer, NEB4 | 5 | |
| 1μl | BSA (10x) | 5 | |
| 0,5 μl | Enzym 1 | 1 | BamHI |
| 0,5 μl | Enzym 2 | - | |
| 3 μl | DNA | 3 |
10 μl total volume
| 4μl | H2O | 20 | |
| 1μl | Buffer, NEB4 | 5 | |
| 1μl | BSA (10x) | 5 | |
| 0,5 μl | Enzym 1 | 1 | EcoRI |
| 0,5 μl | Enzym 2 | 1 | PstI |
| 3 μl | DNA | 3 |
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.
- Picture of the gel
CcaR digested with BamH1 are negativ
- To-do: quickchange (scroll down)
Quickchange
Investigator:Jakob
| Name:
Jakob | Date:
03.08.2011 |
| Continue from Experiment: | |
| Project Name:
Quickchange CcaR | |
PCR-Mixture for one Reaction:
For a 50 µl reaction use
| 32,5µl | H20 | |
| 10µl | 5x Phusion Buffer | |
| 2.5µl | Primer fw | Primer: P12 |
| 2.5µl | Primer dw | Primer: P13 |
| 1µl | dNTPs | |
| 1µl | DNA-Template | DNA: CcaR |
| 0.5 µl | Phusion (add in the end) |
results of transformation from 2nd Aug
Investigators:Julia
transformation worked!
blue light receptor
NAME OF YOUR EXPERIMENT
Investigators:NAME
red light receptor
Colony picking
Investigator:Jakob
Picked some colonies for grow over night
- to-do: Miniprep
Lysis cassette
Quickchange of BBa_K124017 to insert RBS (B0034) and 6bp in front of the first ATG
Primers for Quickchange
- Forward Primer: GGCGGTGATAATGGTTGCTAaagaggagaaaCTAGAGATGCCAGAAAAAACATGAC
- Reverse Primer: GTCATGTTTTTTCTGGCATCTCTAGtttctcctcttTAGCAACCATTATCACCGCC
Quickchange PCR
| Name: Theo
| Date: 03.08.2011 |
| Project Name: Quickchange of BBa_K124017 to insert RBS (B0034) and 6bp in front of the first ATG | |
PCR-Mixture for one Reaction:
For a 50 µl reaction use
| 32,5µl | H20 | Name |
| 10µl | 6x Phusion HF Buffer | |
| 2.5µl | Primer fw | P55 (RBS2LysV2up) (1:10) |
| 2.5µl | Primer dw | P56 (RBS2LysV2dw) (1:10) |
| 1µl | dNTPs | |
| 1µl | DNA-Template | S49 (Composite part -modified K098995+K124017) (->diluted to 5ng/µl) |
| 0.5 µl | Phusion (add in the end) |
What program do you use?
| | |||
| | | | |
| | |||
| |
Digestion of Quickchange with DpnI
| Name: Theo
| Date: 03.08.2011 |
| Continue from Experiment 03.08.2011
Ready Lysis Cassette (Part S49) + RBS | |
| Project Name: Ready Lysis Cassette (Part K124017) + RBS (B0034) | |
| 5μl | Buffer, NEB4 | ||
| 5μl | BSA (10x) | ||
| 2,5 μl | Enzym 1 | DpnI | |
| 37,5 μl | DNA | Quickchange PCR |
50 μl total volume
Incubate for about 1,5h at 37°C + Heat inactivation at 80°C for 20min.
Precipitator
Investigators: Theo
new 3A-assembly with Kan vector to see if pSB1T3 played a negative role in the last 3A-assembly.
Incubation over night at 16°C.
Precipitator
NAME OF YOUR EXPERIMENT
Investigators: NAME
