Team:Groningen/Notebook/Wetwork 23July2012

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Tom and Arjan started with gas chromatography mass spectrometry. Test sample: 5gr. fresh meat incubated @ 37C for 3 houres. For both column (HP5 and CP wax) three runs on each column with different incubation and syringe temperature (1: 80C, 100C 2: 100C, 120C 3: 120C, 140C)
 
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Nisa
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Plasmid isolation pSB1C3-alsT promoter + GFP
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Tom and Arjan started with gas chromatography mass spectrometry. Test sample: 5gr. fresh meat incubated @ 37C for 3 houres. For both column (HP5 and CP wax) three runs on each column with different incubation and syringe temperature (1: 80C, 100C 2: 100C, 120C 3: 120C, 140C)<br>
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<br>
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NanoDrop result (ng/ul):
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<br>
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Nisa<br>
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A1= 102.9
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<br>
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Plasmid isolation pSB1C3-alsT promoter + GFP<br>
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A2= 96,6
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<br>
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NanoDrop result (ng/ul):<br>
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B1= 118,1
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A1= 102.9<br>
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A2= 96,6<br>
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B2= 113,7
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B1= 118,1<br>
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B2= 113,7<br>
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C1= 122,8
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C1= 122,8<br>
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C2= 88 Comment: gel picture OK!<br>
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C2= 88 Comment: gel picture OK!
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D1= 124,9<br>
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D2= 93,3<br>
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D1= 124,9
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<br>
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Result:<br>
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D2= 93,3
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expected size: 3300bp for the pSB1C3 (2079bp)+ alsT (~500bp)+ GFP (800bp)<br>
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<br>
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From the gel picture, only plasmid from colony C2 (promoter alsT300) showed the expected size. The other plasmids seem to be empty, probably due to self ligation.<br>
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Result:
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<br>
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Solution: Check plasmid from other white colony<br>
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expected size: 3300bp for the pSB1C3 (2079bp)+ alsT (~500bp)+ GFP (800bp)
+
<br>
-
 
+
re do transformation. Increase the ratio of plasmid backbone: insert gene<br>
-
From the gel picture, only plasmid from colony C2 (promoter alsT300) showed the expected size. The other plasmids seem to be empty, probably due to self ligation.
+
<br>
-
 
+
<br>
-
Solution: Check plasmid from other white colony
+
Building backbone biobrick for B.subtilis<br>
-
 
+
<br>
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re do transformation. Increase the ratio of plasmid backbone: insert gene
+
plasmid pSac-Cm: integration plasmid, double cross over in SacA region<br>
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+
<br>
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add double terminator, BBa_B0015, and prefix-suffix: XbaI, SpeI, PstI<br>
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Building backbone biobrick for B.subtilis
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<br>
-
 
+
<br>
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plasmid pSac-Cm: integration plasmid, double cross over in SacA region
+
How to? <br>
-
 
+
<br>
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add double terminator, BBa_B0015, and prefix-suffix: XbaI, SpeI, PstI
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Cut backbone with HindIII and EcoRI<br>
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<br>
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Cut BBa_B0015 with HindIII and EcoRI<br>
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How to?  
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<br>
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Now we have separate parts ready to use for ligation into our biobrick device<br>
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Cut backbone with HindIII and EcoRI
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<br>
-
 
+
<br>
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Cut BBa_B0015 with HindIII and EcoRI
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-
 
+
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Now we have separate parts ready to use for ligation into our biobrick device
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<A HREF="https://2012.igem.org/Team:Groningen/Notebook"><FONT COLOR=#ff6700>Back to notebook</FONT></A>
<A HREF="https://2012.igem.org/Team:Groningen/Notebook"><FONT COLOR=#ff6700>Back to notebook</FONT></A>
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<br><br></p></body></html>
{{Template:SponsorsGroningen2012}}
{{Template:SponsorsGroningen2012}}

Latest revision as of 21:20, 25 September 2012







Tom and Arjan started with gas chromatography mass spectrometry. Test sample: 5gr. fresh meat incubated @ 37C for 3 houres. For both column (HP5 and CP wax) three runs on each column with different incubation and syringe temperature (1: 80C, 100C 2: 100C, 120C 3: 120C, 140C)


Nisa

Plasmid isolation pSB1C3-alsT promoter + GFP

NanoDrop result (ng/ul):
A1= 102.9
A2= 96,6
B1= 118,1
B2= 113,7
C1= 122,8
C2= 88 Comment: gel picture OK!
D1= 124,9
D2= 93,3

Result:
expected size: 3300bp for the pSB1C3 (2079bp)+ alsT (~500bp)+ GFP (800bp)

From the gel picture, only plasmid from colony C2 (promoter alsT300) showed the expected size. The other plasmids seem to be empty, probably due to self ligation.

Solution: Check plasmid from other white colony

re do transformation. Increase the ratio of plasmid backbone: insert gene


Building backbone biobrick for B.subtilis

plasmid pSac-Cm: integration plasmid, double cross over in SacA region

add double terminator, BBa_B0015, and prefix-suffix: XbaI, SpeI, PstI


How to?

Cut backbone with HindIII and EcoRI

Cut BBa_B0015 with HindIII and EcoRI

Now we have separate parts ready to use for ligation into our biobrick device


Back to notebook

Our sponsors:

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