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Team:Bielefeld-CeBiTec/Notebook/Journal/rMFC/Aug
From 2014.igem.org
(Difference between revisions)
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</ul> | </ul> | ||
</ul> | </ul> | ||
| + | <ul> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a>of <i>GSU 3274</i> using a gradient (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#pccH_fwd" target="_blank">pccH_fwd</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#pccH_rev" target="_blank">pccH_rev</a>)</li> | ||
| + | <ul> | ||
| + | <li>Annealing temperature: 55 °C appeared as the optimum</li> | ||
| + | <li>Bands as expected (~453 bp)</li> | ||
| + | </ul> | ||
| + | </ul> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a>of <i>GSU 3274</i> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#pccH_fwd" target="_blank">pccH_fwd</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#pccH_rev" target="_blank">pccH_rev</a>)</li> | ||
| + | <ul> | ||
| + | <li>Annealing temperature: 55 °C</li> | ||
| + | <li>Bands as expected (~453 bp)</li> | ||
| + | <li> genomic DNA of <i>G. sulfurreducens</i> as well as the PCR product of the gradient-PCR was used as template</li> | ||
| + | <li>PCR product was <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">purified</a></li> | ||
| + | |||
| + | </ul> | ||
| + | </ul> | ||
| + | |||
| + | |||
Revision as of 20:48, 1 October 2014
 |
August |
 |
- 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 Fum A with electrocompotetent cells
- Colony PCR (VF-Primer, VR-Primer)
- Annealing temperature: 55 °C
- Bands (not) as expected (~... bp)
- BioBrick Assembly (Suffix)
- pR6K
- The pR6K-cassette for the dcuB deletion was purified out of the gel
- ccm
- PCR amplification of the ccm-backbone (pSB1C3_suf_ccm, pSB1C3_pre_ccm)
- Annealing temperature: 65 °C
- Bands as expected (~ 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
