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Team:Bielefeld-CeBiTec/Notebook/Journal/rMFC/Jul
From 2014.igem.org
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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 pSB1C3_<i>oprF</i> </li> | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of pSB1C3_<i>oprF</i> </li> | ||
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| - | |||
| - | |||
| - | |||
| - | |||
</ul> | </ul> | ||
| + | </ul> | ||
| + | <ul> | ||
<li><b> RedET </b></li> | <li><b> RedET </b></li> | ||
<ul> | <ul> | ||
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of RedET plasmid </li> | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PurificationPromega" target="_blank">Plasmid isolation</a> of RedET plasmid </li> | ||
</ul> | </ul> | ||
| + | </ul> | ||
</div> | </div> | ||
</div> | </div> | ||
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<div class="content" style="margin-right:10%; margin-left:10%"> | <div class="content" style="margin-right:10%; margin-left:10%"> | ||
<ul> | <ul> | ||
| - | <li><b> Amplification of the <i>pR6K-cassette</i> and | + | <li><b> Amplification of the <i>pR6K-cassette</i> and the <i>oprF</i> gene for the connection of both</b></li> |
| - | <ul> <li> This week we tried to amplify the <i>pR6K</i>-cassette | + | <ul> <li> This week we tried to amplify the <i>pR6K</i>-cassette and the <i>oprF</i> gene to connect them to each other. |
| + | <ul> | ||
| + | <li><i><b>pR6K</b></i> | ||
<ul> | <ul> | ||
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> of <i>pR6K</i>-cassette (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#pR6K_dcuB_fwd" target="_blank">pR6K_dcuB_fwd</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#pR6K_dcuB_rev" target="_blank">pR6K_dcuB_rev</a>)</li> | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> of <i>pR6K</i>-cassette (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#pR6K_dcuB_fwd" target="_blank">pR6K_dcuB_fwd</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#pR6K_dcuB_rev" target="_blank">pR6K_dcuB_rev</a>)</li> | ||
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</ul> | </ul> | ||
<ul> | <ul> | ||
| - | <li>PCR | + | <li>PCR product <i>pR6K</i> was <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">purified</a> out of the gel</li> |
</ul> | </ul> | ||
| + | </ul> | ||
| + | <ul> | ||
| + | <li><i><b>oprF</b></i></li> | ||
| + | <ul> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification </a> of <i>oprF</i> for integration into chromosome (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#porine_fwd_FRT" target="_blank"> porine_fwd_FRT</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#porine_rev_FRT " target="_blank"> porine_rev_FRT </a>)</li> | ||
| + | <ul> | ||
| + | <li>Annealing temperature: 55 °C</li> | ||
| + | <li>bands are as expected (~1359 bp) </li> | ||
| + | </ul> | ||
| + | <ul> | ||
| + | <li>PCR product <i>oprF</i> was <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">purified</a> out of the gel</li> | ||
| + | </ul> | ||
| + | </ul> | ||
| + | </ul> | ||
| + | <ul> | ||
| + | <li><b> connection of <i>pR6K</i> and <i>oprF</b></i> | ||
<ul> | <ul> | ||
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> to connect the <i>pR6K</i>-cassette and <i>oprF</i> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#porine_fwd_FRT" target="_blank">porine_fwd_FRT</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#pR6K_dcuB_rev" target="_blank">pR6K_dcuB_rev</a>)</li> | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> to connect the <i>pR6K</i>-cassette and <i>oprF</i> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#porine_fwd_FRT" target="_blank">porine_fwd_FRT</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#pR6K_dcuB_rev" target="_blank">pR6K_dcuB_rev</a>)</li> | ||
<ul> | <ul> | ||
<li>Annealing temperature: 55 °C</li> | <li>Annealing temperature: 55 °C</li> | ||
| - | <li>Bands not as expected (~2900 bp)</li> | + | <li>Bands not as expected because of too much template (~2900 bp)</li> |
| + | <li> Next time the <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> was planned with a different usage of primer | ||
| + | <li> additionally a new <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> (as described further up) of <i>pR6K</i>-cassette and a new <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">purification</a> of it and <i>oprF</i> out of the gel was done</li> | ||
</ul> | </ul> | ||
</ul> | </ul> | ||
| - | + | </ul> | |
</div> | </div> | ||
Revision as of 18:38, 30 September 2014
 |
July |
 |
- 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
- RedET
- Plasmid isolation of RedET plasmid
- Amplification of the pR6K-cassette and the oprF gene for the connection of both
- This week we tried to amplify the pR6K-cassette and the oprF gene to connect them to each other.
- pR6K
- 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 product pR6K was purified out of the gel
- 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)
- PCR product oprF was purified out of the gel
- connection of pR6K and oprF
- PCR amplification to connect the pR6K-cassette and oprF (porine_fwd_FRT, pR6K_dcuB_rev)
- Annealing temperature: 55 °C
- Bands not as expected because of too much template (~2900 bp)
- Next time the PCR amplification was planned with a different usage of primer
- additionally a new PCR amplification (as described further up) of pR6K-cassette and a new purification of it and oprF out of the gel was done
- pR6K
