Team:Paris/10 August 2009

From 2009.igem.org

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(→Lab work)
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<div class="experience">
<div class="experience">
GEL!!!
GEL!!!
-
Remigrate to be sure... pFecA seem to be good but not Ompa-Linker
+
*Remigrate to be sure... pFecA seem to be good but not Ompa-Linker
</div>
</div>
 +
<div class="guillaume">
 +
Digestion
 +
</div>
 +
<div class="experience">
 +
*Digestion of P21 (pLac in pSB2K3) with SpeI/PstI (D12) in order to insert D11 RBS-tet (XbaI/PstI)
 +
**20µl of P21
 +
**Vfinal=50µl
 +
**2h at 37°C
 +
</div>
 +
 +
<div class="guillaume">
 +
Purification
 +
</div>
 +
<div class="experience">
 +
*Promega kit
 +
**50µl final
 +
GEL!!!(strange! none solution fall as quickly in the wholes)
 +
</div>
 +
 +
<div class="guillaume">
 +
DO<sub>260</sub>
 +
</div>
 +
<div class="experience">
 +
*D11: RBS-tet : 0,10µg/ml ~730bp
 +
*D12: pSB2K3(pLac) : 0,96µl/ml ~4400bp
 +
</div>
 +
 +
<div class="guillaume">
 +
Try for ligation
 +
</div>
 +
<div class="experience">
 +
*Ligation between [D12] pSB2K3(pLac) SpeI/PstI and [D11] RBS-tet XbaI/PstI. 20µl final
 +
*Ligation 1
 +
**4,5µl RBS-tet XbaI/PstI
 +
**1µl pSB2K3(pLac) SpeI/PstI
 +
**2µl Buffer T4 DNA Lygase 10x
 +
**1µl T4 DNA Lygase
 +
**11,5µl H<sub>20</sub>
 +
<br>
 +
*Ligation 1
 +
**10µl RBS-tet XbaI/PstI
 +
**1µl pSB2K3(pLac) SpeI/PstI
 +
**2µl Buffer T4 DNA Lygase 10x
 +
**1µl T4 DNA Lygase
 +
**6µl H<sub>20</sub>
 +
<br>
 +
*10min at room temperature (~25?)
 +
</div>
 +
<div class="guillaume">
 +
Transformation of the ligation
 +
</div>
 +
<div class="experience">
 +
*As usual, but I forget to done transformation with vector only...so I haven't any control... --!
 +
</div>
<div class="caroline">
<div class="caroline">
Purification
Purification

Revision as of 00:12, 11 August 2009

Contents

NoteBook

June
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July
MTWTFSS
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August
MTWTFSS
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3 4 5 6 7 8 9
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31
September
MTWTFSS
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October
MTWTFSS
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26 27 28 29 30 31

← Yesterday - Tomorrow →

August 10th

Brain work

edit please ^^


Lab work

PCR

  • For A8OmpA-Linker and A9pFecA
  • Duplicate with and without DMSO
    • 1:98°C 1min
    • 2:98°C 10s
    • 3:59°C for A8) / 65°C for A9 30s
    • 4:72°C 8s
    • 5:72°C 10min
    • 6: 4°C ~

Mini-Prep for Plac

Same Protocol as usual ...

Do

Dilution for Plasmids 1,2&3
Plasmid D0(000) D0(266/280) D0(320) Dilution Rule Primer Addition Water Addition
P1 3.10 1.72 0.004 600/310 = 1.93 ~ 2.0 µL 2.45 µL 10.55 µL
P2 1.34 1.34 0.000 600/134 = 4.48 (~ 4.5 µL) 2.45 µL 8.05 µL
P3 0.70 3.39 0.000 600/70 = 8.57 ~ 8.6 µL 2.45µL 3.95µL

Gel Migration

GEL!!!

  • Remigrate to be sure... pFecA seem to be good but not Ompa-Linker

Digestion

  • Digestion of P21 (pLac in pSB2K3) with SpeI/PstI (D12) in order to insert D11 RBS-tet (XbaI/PstI)
    • 20µl of P21
    • Vfinal=50µl
    • 2h at 37°C

Purification

  • Promega kit
    • 50µl final

GEL!!!(strange! none solution fall as quickly in the wholes)

DO260

  • D11: RBS-tet : 0,10µg/ml ~730bp
  • D12: pSB2K3(pLac) : 0,96µl/ml ~4400bp

Try for ligation

  • Ligation between [D12] pSB2K3(pLac) SpeI/PstI and [D11] RBS-tet XbaI/PstI. 20µl final
  • Ligation 1
    • 4,5µl RBS-tet XbaI/PstI
    • 1µl pSB2K3(pLac) SpeI/PstI
    • 2µl Buffer T4 DNA Lygase 10x
    • 1µl T4 DNA Lygase
    • 11,5µl H20


  • Ligation 1
    • 10µl RBS-tet XbaI/PstI
    • 1µl pSB2K3(pLac) SpeI/PstI
    • 2µl Buffer T4 DNA Lygase 10x
    • 1µl T4 DNA Lygase
    • 6µl H20


  • 10min at room temperature (~25?)

Transformation of the ligation

  • As usual, but I forget to done transformation with vector only...so I haven't any control... --!

Purification

purification with promega kits gel purification -> Very good

To do list

Matricule TODO
Luc
Romain
Charlotte
Stoff
Chris
Lisa
Caroline
Souf
Vicard
Pierre
Sylvain
Guillaume


← Yesterday - Tomorrow →

Retrieved from "http://2009.igem.org/Team:Paris/10_August_2009"