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Team:Bielefeld-CeBiTec/Notebook/Journal/rMFC/Aug
From 2014.igem.org
(Difference between revisions)
| Line 546: | Line 546: | ||
<li>next the illegal restriction sites had to be removed</li> | <li>next the illegal restriction sites had to be removed</li> | ||
</ul> | </ul> | ||
| - | + | </ul> | |
| + | </ul> | ||
| + | <br> | ||
| + | <ul><li><b><i>ccm</i></b></li> | ||
| + | <ul> | ||
| + | <li>Eckhardt gel of <i>ccm</i> was performed two times</li> | ||
| + | <ul> | ||
| + | <li> Bands not impeccable to identify (~6230 bp) and therefore a <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">restriction digestion</a> was done</li> | ||
| + | <ul><li>Bands not as expected (~2070 bp and ~6239 bp) </li> | ||
| + | </ul> | ||
| + | </ul> | ||
| + | </ul> | ||
| + | </ul> | ||
| + | <br> | ||
| + | <ul><li><b><i>GSU 3274</i></b></li> | ||
| + | <ul> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#ColonyPCR" target="_blank">Colony PCR</a> of <i>GSU 3274</i> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#VF-Primer" target="_blank">VF-Primer</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#VR-Primer" target="_blank">VR-Primer</a>) | ||
| + | </li> | ||
| + | <ul> | ||
| + | <li>Annealing temperature: 55 °C</li> | ||
| + | <li>Bands as expected (~783 bp)</li> | ||
| + | </ul> | ||
| + | </ul> | ||
| + | <ul> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of <i>GSU 3274</i></li> | ||
| + | </ul> | ||
| + | <ul> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>Pst</i>I</a> and <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>EcoR</i>I</a> </li> | ||
| + | <ul> | ||
| + | <li>Bands as expected (~20170 bp and 453 bp)</li> | ||
| + | </ul> | ||
| + | </ul> | ||
Revision as of 17:55, 5 October 2014
 |
August |
 |
- Deletion of dcuB and integration of oprF into chromosome
- This week we amplified and purified the pR6K-cassette again in order to connect it to oprF
- 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)
- Deletion of dcuB and integration of oprF into chromosome
- pR6K
- The pR6K-cassette for the dcuB deletion was purified out of the gel
- Transformation of pR6K-cassette for the deletion of dcuB with electrocompotetent cells
- pRedET
- Transformation of RedET plasmid with electrocompotetent cells
- dcuB
- 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_J23101_FumA
- BBa_J23102_ Fum A
- BBa_J23104_FumA
- pSB1A2_T7 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
- Sequencing of
- BBa_J23101_FumA
- BBa_J23102_FumA
- BBa_J23104_FumA
- pSB1A2_T7_FumA showed different mutations
- 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
- Deletion of dcuB and integration of oprF into chromosome
- 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)
- PCR amplification to connect the pR6K-cassette and oprF
- Fum A
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (1843 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)
- Deletion of dcuB and integration of oprF into chromosome
- oprF
- NPN-uptake assay for porine verification
- dcuB
- Colony PCR to verify the deletion of dcuB (dcuB_del_kon1, dcuB_del_kon2)
- Annealing temperature: 55°C
- Bands as expected (~4046 bp)
- another Colony PCR to verify the positive colony
- FumA
- BioBrick Assembly (Suffix
- Backbone (digested with SpeI, PstI)
- BBa_J23101
- BBa_J23102
- BBa_J23104
- pSB1A2_T7
- Insert (digested with XbaI, PstI)
- FumA
- Transformation of all contrsucts with electrocompotetent cells
- Colony PCR with all contructs (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~2100 bp) for BBa_J23101_FumA and pSB1A2_T7_FumA
- 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
- ccm
- Eckhardt gel of ccm was performed two times
- Bands not impeccable to identify (~6230 bp) and therefore a restriction digestion was done
- Bands not as expected (~2070 bp and ~6239 bp)
- GSU 3274
- Colony PCR of GSU 3274 (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands as expected (~783 bp)
- Plasmid isolation of GSU 3274
- Restriction digestion with PstI and EcoRI
- Bands as expected (~20170 bp and 453 bp)
