Team:Groningen/Notebook/Wetwork 20July2012

From 2012.igem.org

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Plan for today:
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1. plasmid isolation from E.coli Dh5A
 
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2. ligate alsT promoter and GFP into pSB1C3 and pSac-Cm (B. subtilis backbone)
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3. E.coli transformation for both
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<body><br><br><br>
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<p class="margin">
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Plan for today:<br>
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<br>
 +
1. plasmid isolation from E.coli Dh5A<br>
 +
<br>
 +
2. ligate alsT promoter and GFP into pSB1C3 and pSac-Cm (B. subtilis backbone)<br>
 +
<br>
 +
3. E.coli transformation for both<br>
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<br>
 +
<br>
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1. Plasmid isolation pSB1C3+insert from iGEM Groningen 2010<br>
 +
<br>
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E.coli DH5a strong magenta = insert device OK!<br>
 +
<br>
 +
plasmid concentration: 129,1 ng/ul<br>
 +
<br>
 +
<br>
 +
Purification of PCR product using gel DNA extraction kit<br>
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<br>
 +
diluted in 50ul TE (elution buffer)<br>
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<br>
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concentration: 35,6 ng.ul<br>
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<br>
 +
<br>
 +
Ligation pAlsT-GFP into backbone pSB1C3 (and pSac-Cm done next week 23/07)<br>
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<br>
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Cut parts with restiction enzymes<br>
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<br>
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alsT: EcoRI + SpeI<br>
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<br>
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GFP: XbaI + PstI<br>
 +
<br>
 +
backbone: EcoRI + PstI<br>
 +
<br>
 +
Digestion reaction and subsequently ligation of:<br>
 +
<br>
 +
1) alsT150-GFP-BB<br>
 +
<br>
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2) alsT250-GFP-BB<br>
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<br>
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3) alsT300-GFP-BB<br>
 +
<br>
 +
4) alsT500-GFP-BB<br>
 +
<br>
 +
<br>
 +
3. Transformation of E.coli DH5a<br>
 +
<br>
 +
4x promoter alsT testing devices<br>
 +
<br>
 +
1x negative control<br>
 +
<br>
 +
= 5x LB plate + 15 ug/ml Cm<br>
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<br>
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<br>
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<A HREF="https://2012.igem.org/Team:Groningen/Notebook"><FONT COLOR=#ff6700>Back to notebook</FONT></A>
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</p><br><br>
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</body>
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</html>
-
 
+
{{Template:SponsorsGroningen2012}}
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1. Plasmid isolation pSB1C3+insert from iGEM Groningen 2010
+
-
 
+
-
E.coli DH5a strong magenta = insert device OK!
+
-
 
+
-
plasmid concentration: 129,1 ng/ul
+
-
 
+
-
 
+
-
Purification of PCR product using gel DNA extraction kit
+
-
 
+
-
diluted in 50ul TE (elution buffer)
+
-
 
+
-
concentration: 35,6 ng.ul
+
-
 
+
-
 
+
-
Ligation pAlsT-GFP into backbone pSB1C3 (and pSac-Cm done next week 23/07)
+
-
 
+
-
Cut parts with restiction enzymes
+
-
 
+
-
alsT: EcoRI + SpeI
+
-
 
+
-
GFP: XbaI + PstI
+
-
 
+
-
backbone: EcoRI + PstI
+
-
 
+
-
Digestion reaction and subsequently ligation of:
+
-
1) alsT150-GFP-BB
+
-
 
+
-
2) alsT150-GFP-BB
+
-
 
+
-
3) alsT150-GFP-BB
+
-
 
+
-
4) alsT150-GFP-BB
+

Latest revision as of 21:16, 25 September 2012







Plan for today:

1. plasmid isolation from E.coli Dh5A

2. ligate alsT promoter and GFP into pSB1C3 and pSac-Cm (B. subtilis backbone)

3. E.coli transformation for both


1. Plasmid isolation pSB1C3+insert from iGEM Groningen 2010

E.coli DH5a strong magenta = insert device OK!

plasmid concentration: 129,1 ng/ul


Purification of PCR product using gel DNA extraction kit

diluted in 50ul TE (elution buffer)

concentration: 35,6 ng.ul


Ligation pAlsT-GFP into backbone pSB1C3 (and pSac-Cm done next week 23/07)

Cut parts with restiction enzymes

alsT: EcoRI + SpeI

GFP: XbaI + PstI

backbone: EcoRI + PstI

Digestion reaction and subsequently ligation of:

1) alsT150-GFP-BB

2) alsT250-GFP-BB

3) alsT300-GFP-BB

4) alsT500-GFP-BB


3. Transformation of E.coli DH5a

4x promoter alsT testing devices

1x negative control

= 5x LB plate + 15 ug/ml Cm


Back to notebook



Our sponsors:

Retrieved from "http://2012.igem.org/Team:Groningen/Notebook/Wetwork_20July2012"