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Team:Bielefeld-CeBiTec/Notebook/Journal/rMFC/Jul
From 2014.igem.org
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July |
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- Amplification of pSB1C3 backbone for FumA and FumBCD
- This week we amplified the pSB1C3 backbone of FumA and FumBCD to use it for Gibson Assembly afterwards
- PCR amplification ( pSB1C3_pre_Fum_A, pSB1C3_suf_Fum_A )
- Annealing temperature: 55 °C
- Bands are as expected (2,07 kb)
- PCR amplification ( pSB1C3_pre_Fum_BCD, pSB1C3_suf_Fum_BCD )
- Annealing temperature: 55 °C
- Bands are as expected (2,07 kb)
- PCR purification of backbones
- oprF
- Plasmid isolation of pSB1C3_oprF
- PCR amplification of oprF for integration into chromosome ( porine_fwd_FRT, porine_rev_FRT )
- Annealing temperature: 55 °C
- bands are as expected (~1359 bp)
- RedET
- Plasmid isolation of RedET plasmid
- Amplification of the pR6K-cassette and connection with oprF
- This week we tried to amplify the pR6K-cassette to connect it with the oprF gene
- PCR amplification of pR6K-cassette (pR6K_dcuB_fwd, pR6K_dcuB_rev)
- Annealing temperature: 55 °C
- Bands are as expected (~1600 bp) but slightly visible
- PCR amplification of pR6K-cassette is repeated two times
- PCR products pR6K and oprF were purified out of the gel
- PCR amplification to connect the pR6K-cassette and oprF (porine_fwd_FRT, pR6K_dcuB_rev)
- Annealing temperature: 55 °C
- Bands not as expected (~2900 bp)
