"
Team:Paris/19 August 2009
From 2009.igem.org
(Difference between revisions)
(→Lab work) |
(→Lab work) |
||
| (8 intermediate revisions not shown) | |||
| Line 4: | Line 4: | ||
==NoteBook== | ==NoteBook== | ||
| - | { | + | {{Paris2009_Calendar}} |
| - | + | {{Paris2009_Calendar_Link|18_August_2009|20_August_2009}} | |
| - | + | <center> '''August 19th''' </center> | |
| - | + | ||
| - | + | ||
| - | + | ||
| - | + | ||
<html> | <html> | ||
| Line 18: | Line 14: | ||
<div id="paris_content"> | <div id="paris_content"> | ||
</html> | </html> | ||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
| - | |||
==Lab work== | ==Lab work== | ||
| Line 87: | Line 68: | ||
PCR 2 steps : 72°C 30s elongation | PCR 2 steps : 72°C 30s elongation | ||
| - | Gel verification Ok ( | + | Gel verification Ok (OmpAsignal: weak) |
Purification Ok | Purification Ok | ||
| + | OD : A28=0,82 A29=0,92 | ||
</div> | </div> | ||
| + | <div class="vicard"> | ||
| + | Miniprep send to sequencing. | ||
| + | </div> | ||
| + | <div class="experience"> | ||
| + | Miniprep DO with miniprep diluted 1/100: | ||
| - | + | ClyA colonie 3:1,40 | |
| + | |||
| + | ClyA colonie 7:1,60 | ||
| + | |||
| + | ClyA colonie 8:0,8 | ||
| + | |||
| + | ClyA colonie 10:1,65 | ||
| + | |||
| + | The results were transformed into µg/mL | ||
| + | |||
| + | Mix for sequencing: | ||
| + | |||
| + | {| | ||
| + | |- style="background: #b3d0E4; text-align: center; color:black; " | ||
| + | |width=10%| | ||
| + | |width=10%| Cly A colonie 3 | ||
| + | |width=10%| Cly A colonie 7 | ||
| + | |width=10%| Cly A colonie 8 | ||
| + | |width=10%| Cly A colonie 10 | ||
| + | |||
| + | |||
| + | |- style="background: #E0E9EF; text-align: center;" | ||
| + | |width=10%| Miniprep | ||
| + | |width=10%| 4,29µL | ||
| + | |width=10%| 3,75µL | ||
| + | |width=10%| 7,50µL | ||
| + | |width=10%| 3,64µL | ||
| + | |||
| + | |||
| + | |- style="background: #E0E9EF; text-align: center;" | ||
| + | |width=10%| Oligos | ||
| + | |width=10%| 2,45µL | ||
| + | |width=10%| 2,45µL | ||
| + | |width=10%| 2,45µL | ||
| + | |width=10%| 2,45µL | ||
| + | |||
| + | |- style="background: #E0E9EF; text-align: center;" | ||
| + | |width=10%| H2O | ||
| + | |width=10%| 8,26µL | ||
| + | |width=10%| 8,80µL | ||
| + | |width=10%| 5,05µL | ||
| + | |width=10%| 8,91µL | ||
| + | |||
| + | |} | ||
</div> | </div> | ||
| - | <div | + | <div class="vicard"> |
| + | RFP PCR | ||
</div> | </div> | ||
| - | <div | + | <div class="experience"> |
| - | + | ||
| - | + | RFP PCR using PSB1A3 and: | |
| - | + | -O57+O58==>A30=RFP Nter | |
| + | |||
| + | -O59+058==>A31=RFP | ||
| + | |||
| + | -O59+O60==>A33=RFP Cter | ||
| + | |||
| + | Mix: | ||
| + | |||
| + | H2O:32,5µL | ||
| + | |||
| + | Buffer 5x:10µL | ||
| + | |||
| + | dNTP:1µL | ||
| + | |||
| + | OF(1/10):2,5µL | ||
| + | |||
| + | OR(1/10):2,5µL | ||
| + | |||
| + | Matrice(PSB1A3 1/10):1µL | ||
| + | |||
| + | Phusiong:0,5µL | ||
| + | |||
| + | '''PCR Check''' | ||
| + | |||
| + | /100pb/A30/A31/A33/1kb | ||
| + | |||
| + | </div> | ||
| + | |||
| + | <div class="vicard"> | ||
| + | Digestion | ||
| + | </div> | ||
| + | <div class="experience"> | ||
| + | |||
| + | Digestion PSB1A3 in XP | ||
| + | |||
| + | </div> | ||
| + | {{Paris2009_Calendar_Link|18_August_2009|20_August_2009}} | ||
Latest revision as of 12:06, 10 October 2009
NoteBook
|
|
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Lab work
Sequencing w/ Bobode
5 clones pLac/RBS-TetR have to be sequenced
Clone 1, 2, 6, 7, 8 used (because of their high DNA concentration)
Transformation w/ Bobode
pSB1A3-TolRII ligation was transformed into P10 chemical competent cells, using the "heat shock" protocol
Glycerol stock w/ Charlie
Glycerol stock of :
S39 - pINTE3 (Col E3, AmpR)
S40 - pINTg3p (g3p Term, AmpR)
S41 - pINK2(TolRII, AmpR)
S42 - pTA (pTet-Col A, AmpR)
A28 A29
A28 OmpA signal (ex A11)
A29 FecR mutant
PCR 2 steps : 72°C 30s elongation
Gel verification Ok (OmpAsignal: weak)
Purification Ok
OD : A28=0,82 A29=0,92
Miniprep send to sequencing.
Miniprep DO with miniprep diluted 1/100:
ClyA colonie 3:1,40
ClyA colonie 7:1,60
ClyA colonie 8:0,8
ClyA colonie 10:1,65
The results were transformed into µg/mL
Mix for sequencing:
| Cly A colonie 3 | Cly A colonie 7 | Cly A colonie 8 | Cly A colonie 10
| |
| Miniprep | 4,29µL | 3,75µL | 7,50µL | 3,64µL
|
| Oligos | 2,45µL | 2,45µL | 2,45µL | 2,45µL |
| H2O | 8,26µL | 8,80µL | 5,05µL | 8,91µL |
RFP PCR
RFP PCR using PSB1A3 and:
-O57+O58==>A30=RFP Nter
-O59+058==>A31=RFP
-O59+O60==>A33=RFP Cter
Mix:
H2O:32,5µL
Buffer 5x:10µL
dNTP:1µL
OF(1/10):2,5µL
OR(1/10):2,5µL
Matrice(PSB1A3 1/10):1µL
Phusiong:0,5µL
PCR Check
/100pb/A30/A31/A33/1kb
Digestion
Digestion PSB1A3 in XP
