Joyce
PCRs with different samples, scheme:
Samples PhybB: DNA TG1 3×TG1 Colony DH5alpha 3× colonies DH5alpha
pfu 10× buffer: 5μl 15μl 5μl 15μl
dNTPs 10mM: 1μl 3μl 1μl 3μl
Forward Biobrick primer: 1μl 3μl 1μl 3μl
Reverse Biobrick primer: 1μl 3μl 1μl 3μl
Pfu DNA polymerase: 1μl 3μl 1μl 3μl
MQ water: 41μl 123μl 41μl 123μl
PBAD and PlasI PCRs:
pfu 10× buffer: 5μl
dNTPs 10mM: 1μl
Forward Biobrick primer: 1μl
Reverse Biobrick primer: 1μl
Pfu DNA polymerase: 1μl
MQ water: 41μl
PCR conditions:
Preheated lid: 111°C
Denaturation: 95°C for 3 min.
Cycle 33×:
denaturation: 95°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 samples on a 1% agarose gel
Clean up the DNA samples
work on human practices
Jakub
Week 25:
- cloning PlasB promoter into plasmid with reverse double terminator
- cloning reversed pBAD/AraC promoter into plasmid with double terminator
Christoph
- Repeated cloning of some cI variants into pSB1K3+rev DT.
- Struggling with the PCR to obtain the right PCR products for some cI variants.
"
