IGEM:Groningen/Notebook/iGEM 2011/2011/06/14

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(Autocreate 2011/06/14 Entry for IGEM:Groningen/Notebook/iGEM_2011)
Current revision (05:38, 21 September 2011) (view source)
(Entry title)
 
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==Entry title==
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==14-6-11==
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* Insert content here...
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<br>
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<br> Checking transformants/ testing whether DNA purification from gel went well and is functional in cloning:
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<br> PlasB from gel+ vector from gel: no colonies, maybe 1 or 2
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<br> PlasB from gel+ vector no gel: Lots of colonies, might be a lot of self ligation, but the self ligation control is negative...
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<br> PBADaraC from gel+ vector from gel: no colonies, just a few but it looks like contamination?
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<br> PBADaraC no gel+  vector gel: much colonies
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<br> PBADaraC gel + vector no gel: lots of colonies
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<br>
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<br> Grow colonies , 3 colonies per different construct for plasmid prepping
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<br>
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<br> Colony PCR
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<br> Taq 10× buffer: 40μl
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<br> dNTPs 10mM: 8μl
 +
<br> Forward Biobrick primer: 8μl
 +
<br> Reverse Biobrick primer: 8μl
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<br> Taq DNA polymerase: 2μl
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<br> MQ water: 334μl
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<br>
 +
<br> PCR conditions:
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<br> Preheated lid: 111°C
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<br> Denaturation: 94°C for 10 min.
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<br> Cycle 33×:
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<br>  denaturation: 94°C for 30s.
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<br>  annealing:    60°C for 30s.
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<br>  Extension:    72°C for 2,5 min.
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<br> Final extension: 72°C for 10 min.
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<br> Store infinite at 4°C
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<br>
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<br> Analyse on a 1% TBE agarose gel.
 +
<br>
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14-6-11



Checking transformants/ testing whether DNA purification from gel went well and is functional in cloning:
PlasB from gel+ vector from gel: no colonies, maybe 1 or 2
PlasB from gel+ vector no gel: Lots of colonies, might be a lot of self ligation, but the self ligation control is negative...
PBADaraC from gel+ vector from gel: no colonies, just a few but it looks like contamination?
PBADaraC no gel+ vector gel: much colonies
PBADaraC gel + vector no gel: lots of colonies

Grow colonies , 3 colonies per different construct for plasmid prepping

Colony PCR
Taq 10× buffer: 40μl
dNTPs 10mM: 8μl
Forward Biobrick primer: 8μl
Reverse Biobrick primer: 8μl
Taq DNA polymerase: 2μl
MQ water: 334μl

PCR conditions:
Preheated lid: 111°C
Denaturation: 94°C for 10 min.
Cycle 33×:
denaturation: 94°C for 30s.
annealing: 60°C for 30s.
Extension: 72°C for 2,5 min.
Final extension: 72°C for 10 min.
Store infinite at 4°C

Analyse on a 1% TBE agarose gel.

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