Purification of proteins: Z-alpha and Z-omegaPiotr, Emilia, Weronika
- pET15b+Z_alpha and pET15b+Z_omega in Rosetta strain from overnight culture inoculated in fresh LB and cultured until OD=0,5.
- Cultures induced with different concentrations of IPTG in 22°C and 37°C.
- Samples were collected twice: after 3h and next day (samples were centrifuged and frozen).
Michał K.
Separate transformant colonies (tranformation of ligation of pACYC177+OmpA_alpha and omega_ΔA from previous day) inoculated to liquid LB with kanamycin.
Cloning of truncated fragment of protein A (ΔA)
Michał K.
- Optimization of PCR to obtain truncated fragment of protein A DNA.
Primers:
AL+SacI
AP+NotI
Template DNA: pDRIVE-TapTag
Elongation time: 30s
- Optimization of annealing temperature (gradient from 55°C to 75°C). Fig. 1.
- Optimization of number of cycles(15, 20, 25, 30, 35). Fig. 2.
- PCR to obtain truncated A protein DNA fragment.
Primers:
AL+SacI
AP+NotI
Template DNA: pDRIVE-TapTag
Elongation time: 30s
Annealing temperature: 60°C
20 cycles
- Gel electrophoresis and gel-out of 250 bp band. Fig. 3.
- Digest of isolated PCR product, pACYC177+OmpA_alpha and pACYC177+OmpA_omega with NotI and SacI (BamHI buffer). pACYC177 vectors were also dephosphorylated.
- Clean-up of digest reaction.
- Gel electrophoresis for estimation of DNA concentration.
- Overnight ligation: pACYC177+OmpA_alpha + ΔA and pACYC177+OmpA_omega + ΔA.
 Fig. 1. Gradient PCR to obtain truncated protein A (temperatures: 55-75°C)
1. Marker
2. 50°C
3. 55°C
4. 60°C
5. 65°C
6. 70°C
7. 75°C
 Fig. 2. PCR to obtain truncated protein A (various number of cycles)
1. Marker
2. 15 cycles
3. 20 cycles
4. 25 cycles
5. 30 cycles
6. 35 cycles
 Fig. 3. PCR amplified truncated protein A
1. Marker
2. PCR product (deltaA), temperature of annealing = 60°C, 20 cycles
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