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Team:Bielefeld-CeBiTec/Notebook/Journal/rMFC/Aug
From 2014.igem.org
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| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1C3_<i>frd</i></li> | ||
| + | </ul> | ||
| + | <ul> | ||
| + | <li>sequencing of pSB1C3_<i>frd</i> confirmed the correct construct</li> | ||
| + | </ul> | ||
| + | <ul> | ||
| + | <ul> | ||
| + | <li>next the illegal restriction sites had to be removed</li> | ||
| + | </ul> | ||
Revision as of 11:31, 5 October 2014
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August |
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- pR6K
- PCR amplification of pR6K-cassette
- Plasmid isolation of pR6K-cassette and Purification out of the gel
- Connection of the pR6K-cassette and oprF
- PCR amplification to connect the pR6K-cassette and oprF
- Bands as expected (~3300 bp)
- PCR product was purified out of the gel
- Fum A
- Plasmid isolation of Fum A
- BioBrick Assembly (Suffix)
- Transformation of pSB1C3_T7_Fum A with electrocompotetent cells
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~2130 bp)
- BioBrick Assembly (Suffix)
- pR6K
- The pR6K-cassette for the dcuB deletion was purified out of the gel
- dcuB
- Transformation of RedET plasmid with electrocompotetent cells
- Transformation of pR6K-cassette for the deletion of dcuB with electrocompotetent cells
- Colony PCR to verify the deletion of dcuB (dcuB_del_kon1, dcuB_del_kon2)
- Annealing temperature: 55 °C
- Bands not as expected (~2390 bp)
- Fum A
- Transformation of BBa_J23102_ Fum A with electrocompotetent cells
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Band as expected (~2200 bp)
- Plasmid isolation of BBa_J23102_ Fum A
- Fum BCD
- PCR amplification of Fum BCD-backbone (pSB1C3_pre_Fum_BCD , pSB1C3_pre_Fum_BCD)
- Annealing temperature: 55 °C
- Bands as expected (2070 bp)
- The Fum BCD backbone was purified out of the gel
- PCR amplification of Fum BCD (Fum_BCD_fwd , Fum_BCD_rev)
- Annealing temperature: 55 °C
- Bands not as expected (1579 bp)
- ccm
- PCR amplification of the ccm-backbone (pSB1C3_suf_ccm, pSB1C3_pre_ccm)
- Annealing temperature: 65 °C
- Bands as expected (~2070 bp) but slightly visible because of inappropriate annealing temperature
- PCR amplification was repeated with an annealing temperature of 55 °C
- Both PCR products were purified out of the gel
- PCR amplification of ccm-cassette using a gradient (ccm_fwd, ccm_rev)
- Annealing temperature: 61 °C appeared as the optimum
- Bands as expected (~6281 bp)
- PCR amplification of ccm-cassette (ccm_fwd, ccm_rev)
- Annealing temperature: 61 °C
- Bands as expected (~6281 bp)
- PCR product was purified
- GSU 3274
- PCR amplification of GSU 3274-backbone (pSB1C3_pre_pccH, pSB1C3_suf_pccH)
- Annealing temperature: 65 °C
- Bands not as expected (~2070 bp) because of inappropriate annealing temperature
- PCR amplification was repeated with an annealing temperature of 55 °C
- Bands as expected (~2070 bp)
- PCR amplificationof GSU 3274 using a gradient (pccH_fwd, pccH_rev)
- Annealing temperature: 55 °C appeared as the optimum
- Bands as expected (~453 bp)
- PCR amplificationof GSU 3274 (pccH_fwd, pccH_rev)
- Annealing temperature: 55 °C
- Bands as expected (~453 bp)
- genomic DNA of G. sulfurreducens as well as the PCR product of the gradient-PCR was used as template
- PCR product was purified
- Fum A
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (1843 bp)
- Connection of pR6K-cassette and oprF
- PCR amplification to connect the pR6K-cassette and oprF
- Bands as expected (~3300 bp)
- PCR product was purified out of the gel
- Colony PCR (dcuB_del_kon1, dcuB_del_kon2)
- Annealing temperature: 55 °C
- Bands not as expected (~4046 bp)
- frd (E. coli)
- PCR amplification (T7_frd_Ec_fwd, T7_frd_Ec_rev)
- Annealing temperature: 55 °C
- Bands as expected (~3300 bp)
- Additionally there were undefined bands at ~1200 bp
- illiegal restriction sites were not removed yet
- Gibson Assembly with frd (E. coli) and pSB1C3
- Transformation with electrocompotetent cells of pSB1C3_frd
- Colony PCR of pSB1C3_frd (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands not as expected (~3600 bp)
- ccm
- DpnI-digest of ccm-backbone
- Gibson Assembly with ccm and pSB1C3
- Transformation with electrocompotetent cells of pSB1C3_ccm
- Colony PCR of pSB1C3_ccm (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands not as expected (~6611 bp) probably because it was not completely amplified because of its length
- To check whether one of the colonies is positiv a restriction digestion was done
- Restriction digestion with EcoRI and PstI
- Bands not as expected (~2070 bp and 6281 bp)
- GSU 3274
- DpnI-digest of GSU 3274-backbone
- Gibson Assembly with GSU 3274 and pSB1C3
- Transformation with electrocompotetent cells of pSB1C3_GSU 3274
- Colony PCR of pSB1C3_GSU 3274 (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~783 bp)
- Restriction digestion with EcoRI and PstI
- Bands not as expected (~2070 bp and 453 bp)
- frd (E. coli)
- DpnI digest of Gibson-Assembly of frd (E. coli) to remove the template
- Transformation of Gibson-Assembly with electrocompotetent cells
- Colony PCR of pSB1C3_frd (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~3600 bp) but there were several other bands too
- Therefore another Transformation with electrocompotetent cells was done
- Colony PCR of pSB1C3_frd (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands not as expected (~ 3600 bp)
- PCR amplification of frd backbone (pSB1C3_frd_pre, pSB1C3_frd_suf)
- Annealing temperature: 55 °C
- Bands as expected (~ 2070 bp)
- frd backbone was purified out of the gel
- Gibson Assembly with frd and pSB1C3
- Transformation of pSB1C3_frd with chemocompetent cells
- Colony PCR of pSB1C3_frd (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~ 3600 bp)
- Restriction digestion with EcoRI and PstI
- Bands as expected (~2070 bp and 3300 bp)
- Plasmid isolation of pSB1C3_frd
- sequencing of pSB1C3_frd confirmed the correct construct
- next the illegal restriction sites had to be removed
