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Team:Bielefeld-CeBiTec/Notebook/Journal/rMFC/Aug
From 2014.igem.org
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</ul> | </ul> | ||
<li>Insert (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>Xba</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li> | <li>Insert (digested with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionEnzymes" target="_blank"><i>Xba</i>I</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#RestrictionDigestion" target="_blank"><i>Pst</i>I</a>)</li> | ||
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<ul> | <ul> | ||
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of <a href="http://parts.igem.org/Part:BBa_J23102" target="_blank">BBa_J23102</a>_ <i>Fum A </i> </li> | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of <a href="http://parts.igem.org/Part:BBa_J23102" target="_blank">BBa_J23102</a>_ <i>Fum A </i> </li> | ||
| + | </ul> | ||
| + | </ul> | ||
| + | <ul> | ||
| + | <li><b><i>Fum BCD</i></b></li> | ||
| + | <ul> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> of <i>Fum BCD</i>-backbone (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#pSB1C3_pre_Fum_BCD" target="_blank">pSB1C3_pre_Fum_BCD </a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#pSB1C3_suf_Fum_BCD " target="_blank">pSB1C3_pre_Fum_BCD</a>)</li> | ||
| + | <ul> | ||
| + | <li>Annealing temperature: 55 °C</li> | ||
| + | <li>Bands as expected (~ bp)</li> | ||
| + | </ul> | ||
| + | </ul> | ||
| + | <ul> | ||
| + | <li> The <i>Fum BCD</i> backbone was <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">purified</a> out of the gel</li> | ||
| + | </ul> | ||
| + | <ul> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> of <i>Fum BCD</i> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Fum_BCD_fwd" target="_blank">Fum_BCD_fwd </a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#Fum_BCD_rev " target="_blank">Fum_BCD_rev</a>)</li> | ||
| + | <ul> | ||
| + | <li>Annealing temperature: 55 °C</li> | ||
| + | <li>Bands not as expected (~ bp)</li> | ||
| + | </ul> | ||
</ul> | </ul> | ||
</ul> | </ul> | ||
Revision as of 22:03, 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 pSB1C3_T7_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
- 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 (~ 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 (~ bp)
- 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
