IGEM:Groningen/Notebook/iGEM 2011/2011/06/08

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==Entry title==
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==08-06-11==
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* Insert content here...
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<br>
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<br> Biobricks were checked (by sequencing)
 +
<br>
 +
<br>        Transform:      Digestion analysis:    Sequencing:
 +
<br> PlasB:      OK                  OK
 +
<br> RFP:        OK                  OK
 +
<br> cILVA:      OK                  OK
 +
<br> DT:        OK                  OK
 +
<br> rev DT:    OK                  OK
 +
<br> PBADaraC:  OK                  OK
 +
<br> PcI:        -                  -
 +
<br> GFP-LVA:    -                  -
 +
<br> RBS-GFP-DT: OK                  OK
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<br>
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<br> Plan: Digestion of RBS-GFP-DT with EcoRI and XbaI
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<br> PlasB and PBADaraC with EcoRI and SpeI
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<br> isolate both of gel and use also normal PBADaraC sample for ligation:
 +
<br>
 +
<br> Digestion vector:
 +
<br> 2μl FD buffer
 +
<br> 0.5μl EcoRI
 +
<br> 0.5μl XbaI
 +
<br> 7μl MQ water
 +
<br> 10μl mix+ 10μl sample
 +
<br>
 +
<br> Digestion mix insert:
 +
<br> 14μl FD buffer
 +
<br> 3.5μl EcoRI
 +
<br> 3.5μl SpeI
 +
<br> 49μl MQ water
 +
<br>
 +
<br> Digest for 45 min at 37 degrees
 +
<br>
 +
<br> Clean up of the DNA with the PCR purification kit
 +
<br>
 +
<br> Ligation:
 +
<br> 7μl insert
 +
<br> 3μl plasmid
 +
<br> 2μl T4 DNA ligase buffer
 +
<br> 1μl T4 DNA ligase
 +
<br> 7μl MQ water
 +
<br>
 +
<br> Ligate for 2hours at room temperature and afterwards,ligate overnight
 +
<br>
 +
 
 +
 
 +
 
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08-06-11



Biobricks were checked (by sequencing)

Transform: Digestion analysis: Sequencing:
PlasB: OK OK
RFP: OK OK
cILVA: OK OK
DT: OK OK
rev DT: OK OK
PBADaraC: OK OK
PcI: - -
GFP-LVA: - -
RBS-GFP-DT: OK OK

Plan: Digestion of RBS-GFP-DT with EcoRI and XbaI
PlasB and PBADaraC with EcoRI and SpeI
isolate both of gel and use also normal PBADaraC sample for ligation:

Digestion vector:
2μl FD buffer
0.5μl EcoRI
0.5μl XbaI
7μl MQ water
10μl mix+ 10μl sample

Digestion mix insert:
14μl FD buffer
3.5μl EcoRI
3.5μl SpeI
49μl MQ water

Digest for 45 min at 37 degrees

Clean up of the DNA with the PCR purification kit

Ligation:
7μl insert
3μl plasmid
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
7μl MQ water

Ligate for 2hours at room temperature and afterwards,ligate overnight




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