INSA-Lyon/22 September 2010
From 2010.igem.org
(New page: <!-- {{INSA-Lyon_style}} --> {{INSA-Lyon/stylegroningen}} {{INSA-Lyon/header}} {{INSA-Lyon/maincontent}} {{INSA-Lyon/menugauche}} <html xmlns="http://www.w3.org/1999/xhtml" dir="ltr" ...) |
|||
(3 intermediate revisions not shown) | |||
Line 1: | Line 1: | ||
<!-- {{INSA-Lyon_style}} --> | <!-- {{INSA-Lyon_style}} --> | ||
- | |||
{{INSA-Lyon/stylegroningen}} | {{INSA-Lyon/stylegroningen}} | ||
- | |||
{{INSA-Lyon/header}} | {{INSA-Lyon/header}} | ||
- | |||
{{INSA-Lyon/maincontent}} | {{INSA-Lyon/maincontent}} | ||
- | |||
{{INSA-Lyon/menugauche}} | {{INSA-Lyon/menugauche}} | ||
+ | {{INSA-Lyon/menunotebook}} | ||
<html xmlns="http://www.w3.org/1999/xhtml" dir="ltr" lang="en-US" xml:lang="en"> | <html xmlns="http://www.w3.org/1999/xhtml" dir="ltr" lang="en-US" xml:lang="en"> | ||
Line 47: | Line 44: | ||
</script> | </script> | ||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
- | |||
Line 117: | Line 79: | ||
<p>DNA extraction of the clone of the ligation from the 21/09 of phaA/phaB </p><br> | <p>DNA extraction of the clone of the ligation from the 21/09 of phaA/phaB </p><br> | ||
- | <p>Agarose gel | + | <p>Agarose gel electrophoresis for weight verification : <br> |
<ol id="list_extra"> | <ol id="list_extra"> | ||
<li>1F/24A : the clone (mise en )collection is OK</li> | <li>1F/24A : the clone (mise en )collection is OK</li> | ||
Line 129: | Line 91: | ||
</ol><br> | </ol><br> | ||
</p> | </p> | ||
- | <p>Ligation overnight of 8C/14K and | + | <p>Ligation overnight of : <br> |
- | < | + | <ol id="list_extra"> |
- | <p>Measure of fluorescence and DO of the liquid | + | <li>8C/14K : one with only 14K from gel purification and one with the 8C and 14K from a gel purification</li> |
+ | <li>1F/24A/14K : one ligation</li> | ||
+ | </ol></p><br> | ||
+ | <p>Measure of fluorescence and DO of the liquid culture of PHL1273 of the 20/09. Start of 6 new cultures : <br> | ||
<ol id="list_extra"> | <ol id="list_extra"> | ||
<li> 3 in a water bath at 35°C, shaking speed 100 rpm</li> | <li> 3 in a water bath at 35°C, shaking speed 100 rpm</li> | ||
<li> 3 in a water bath at 36°C, shaking speed 100 rpm</li> | <li> 3 in a water bath at 36°C, shaking speed 100 rpm</li> | ||
- | </ol><br></p> | + | </ol><br></p><br> |
+ | <p>Gel purification on vial 3 of ompR234 PCR (DNA dilution 1/10): 20µl are deposited in a lane. 100mg agarose (in 200µl NT) are extracted with NucleoSpin ExtractII kit (Macherey-Nagel). Elution was done in 50µl, and sample was stored at -20°C.</p><br> | ||
+ | <p>5 liquid cultures in LB medium (5ml) supplemented with corresponding antibiotic: 14K (Amp), I0500 (Kan), 1F/24A (Tet) and 18A/24A (Cm), and PHL818 (Tet), 37°C overnight.</p> | ||
Latest revision as of 20:03, 25 October 2010
September 22nd
DNA extraction of the clone of the ligation from the 21/09 of phaA/phaB
Agarose gel electrophoresis for weight verification :
- 1F/24A : the clone (mise en )collection is OK
- Curli/22B : the clone (mise en )collection is OK
- 8C/14K : problem with the extraction (no trace of DNA on the gel)
Digestion and purification on agarose gel:
- 8C, 1F/24A : with S+P
- 14K with X+P
Ligation overnight of :
- 8C/14K : one with only 14K from gel purification and one with the 8C and 14K from a gel purification
- 1F/24A/14K : one ligation
Measure of fluorescence and DO of the liquid culture of PHL1273 of the 20/09. Start of 6 new cultures :
- 3 in a water bath at 35°C, shaking speed 100 rpm
- 3 in a water bath at 36°C, shaking speed 100 rpm
Gel purification on vial 3 of ompR234 PCR (DNA dilution 1/10): 20µl are deposited in a lane. 100mg agarose (in 200µl NT) are extracted with NucleoSpin ExtractII kit (Macherey-Nagel). Elution was done in 50µl, and sample was stored at -20°C.
5 liquid cultures in LB medium (5ml) supplemented with corresponding antibiotic: 14K (Amp), I0500 (Kan), 1F/24A (Tet) and 18A/24A (Cm), and PHL818 (Tet), 37°C overnight.
|
|
|
|
|