IGEM:Groningen/Notebook/iGEM 2011/2011/08/11: Difference between revisions

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==Entry title==
==11-8-11==
* Insert content here...
<br> Plasmid prep of overnight culture PBAD-pSB1C3 colonies 1 and 2
<br> Measure DNA concentration: 32 ng/microliter
<br> Samples were send for sequencing and glycerol stocks were made
<br>
<br> colony PCR of PBAD-pSB1C3:
<br> Composition master mix:
<br> 10× Taq buffer: 40μl
<br> dNTPs 10mM: 8μl
<br> MgCl2: 24μl
<br> Forward biobrickvector primer: 8μl
<br> Reverse biobrickvector primer: 8μl
<br> Taq polymerase: 2μl
<br> MQ water: 310μl
<br>
<br> PCR conditions:
<br> Pre heated lid: 111°C
<br> Denaturation: 94°C for 10min
<br> Cycle (33×)
<br>  Denaturation: 94°C for 30s
<br>  Annealing: 60°C for 30s
<br>  Extenstion: 72°C for 30s
<br> Final extension: 72°C for 10min
<br> Store infinite at 4°C
<br> Analyse PCR samples on a 1% agarosegel
<br>
<br> dsDNA was digested:
<br> for taRNA fragment:
<br> 5μl dsDNA plasmid
<br> 1μl SacI
<br> 1μl BamHI
<br> 2μl FD buffer
<br> 11μl MQ
<br>
<br> dsDNA was digested:
<br> for crRNA fragment:
<br> 5μl dsDNA plasmid
<br> 1μl BamHI
<br> 1μl SalI
<br> 2μl FD buffer
<br> 11μl MQ
<br>
<br> dsDNA was digested:
<br> for PRM fragment:
<br> 5μl dsDNA plasmid
<br> 1μl SalI
<br> 1μl HindIII
<br> 2μl FD buffer
<br> 11μl MQ
<br>
<br> Isolate from 1% agarose gel with the agarose extraction kit
<br> PCR overnight for taRNA fragment:
<br> 10× pfu+ MgSO4 buffer: 5μl
<br> dNTPs: 1μl
<br> F3: 1μl
<br> R4: 1μl
<br> pfu: 1μl
<br> DNA template: 5μl
<br> 36μl MQ
<br>
<br> PCR conditions:
<br> Pre heated lid: 111°C
<br> Denaturation: 95°C for 3min
<br> Cycle (35×)
<br>  Denaturation: 95°C for 30s
<br>  Annealing: 53°C for 30s
<br>  Extenstion: 72°C for 1min
<br> Final extension: 72°C for 10min
<br> Store infinite at 4°C
<br>




Revision as of 08:23, 11 August 2011

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11-8-11


Plasmid prep of overnight culture PBAD-pSB1C3 colonies 1 and 2
Measure DNA concentration: 32 ng/microliter
Samples were send for sequencing and glycerol stocks were made

colony PCR of PBAD-pSB1C3:
Composition master mix:
10× Taq buffer: 40μl
dNTPs 10mM: 8μl
MgCl2: 24μl
Forward biobrickvector primer: 8μl
Reverse biobrickvector primer: 8μl
Taq polymerase: 2μl
MQ water: 310μl

PCR conditions:
Pre heated lid: 111°C
Denaturation: 94°C for 10min
Cycle (33×)
Denaturation: 94°C for 30s
Annealing: 60°C for 30s
Extenstion: 72°C for 30s
Final extension: 72°C for 10min
Store infinite at 4°C
Analyse PCR samples on a 1% agarosegel

dsDNA was digested:
for taRNA fragment:
5μl dsDNA plasmid
1μl SacI
1μl BamHI
2μl FD buffer
11μl MQ

dsDNA was digested:
for crRNA fragment:
5μl dsDNA plasmid
1μl BamHI
1μl SalI
2μl FD buffer
11μl MQ

dsDNA was digested:
for PRM fragment:
5μl dsDNA plasmid
1μl SalI
1μl HindIII
2μl FD buffer
11μl MQ

Isolate from 1% agarose gel with the agarose extraction kit
PCR overnight for taRNA fragment:
10× pfu+ MgSO4 buffer: 5μl
dNTPs: 1μl
F3: 1μl
R4: 1μl
pfu: 1μl
DNA template: 5μl
36μl MQ

PCR conditions:
Pre heated lid: 111°C
Denaturation: 95°C for 3min
Cycle (35×)
Denaturation: 95°C for 30s
Annealing: 53°C for 30s
Extenstion: 72°C for 1min
Final extension: 72°C for 10min
Store infinite at 4°C


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