Team:Warsaw/Calendar-Main/31 July 2008

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Checking if omp_omega_A_alpha gives ampicillin resistance
Piotr

Inoculation of omp_omega_A_alpha into various IPTG concentrations: 0, 0.1, 0.25, 0.5, 0.75, 1 mmol/mL

1. Optimization of PCR to obtain truncated fragment of protein A DNA

Primers: AL+SacI AP+NotI

Elongation time: 30s

- Optimization of annealing temperature (gradient from 55°C to 75°C)
- Optimization of number of cycles(15, 20, 25, 30, 35)

2. PCR to obtain truncated A protein DNA fragment

Primers: AL+SacI AP+NotI

Elongation time: 30s

Annealing temperature: 60°C

20 cycles

3. Gel electrophoresis and isolation of 250 bp band.
4. Digest of isolated PCR product, pACYC177+OmpA_alpha and pACYC177+OmpA_omega with NotI and SacI.
5. Clean-up of digestion reaction.
6. Gel electrophoresis for estimation of DNA concentration.
7. Overnight ligation: pACYC177+OmpA_alpha + deltaA and pACYC177+OmpA_omega + deltaA.

Checking if degradation of fusion with OmpA is a result of Top10 proteases' activity (lon, iompt)

Piotr:
Inoculation of OmpA_omega_A_alpha and OmpA_A_alpha with and without inductor (0,5 mm/mL IPTG) in E. coli Rosetta strain.

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+<h3>Checking if omp_omega_A_alpha gives ampicillin resistance<br>
+Piotr</h3>
+<ol><li>Inoculation of omp_omega_A_alpha into various IPTG concentrations: 0, 0.1, 0.25, 0.5, 0.75, 1 mmol/mL</li></ol>
 <center><h4><p>1. Optimization of PCR to obtain truncated fragment of protein A DNA </center>
 </h4>