IGEM:Groningen/Notebook/iGEM 2011/2011/08/11

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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× taq buffer: 5μl
dNTPs: 1μl
F3: 1μl
R4: 1μl
pfu: 0.04μl
taq: 0.25μl
DNA template: 5μl
33.71μl MQ

PCR conditions:
Pre heated lid: 111°C
Denaturation: 94°C for 10min
Cycle (35×)
Denaturation: 94°C for 30s
Annealing: 53°C for 30s
Extenstion: 72°C for 1min
Final extension: 72°C for 10min
Store infinite at 4°C