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Team:Bielefeld-CeBiTec/Notebook/Journal/rMFC/Jul
From 2014.igem.org
(Difference between revisions)
| Line 91: | Line 91: | ||
</ul> | </ul> | ||
<ul> | <ul> | ||
| - | <li><b> RedET </b></li> | + | <li><b> RedET plasmid </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> | ||
| Line 114: | Line 114: | ||
<ul> | <ul> | ||
<li><b> Amplification of the <i>pR6K-cassette</i> and the <i>oprF</i> gene for the connection of both</b></li> | <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 and the <i>oprF</i> gene to connect them to each other. | + | <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. Additionally we transform the RedET plasmid into the <i> E. coli </i> strain KRX. |
<ul> | <ul> | ||
<li><i><b>pR6K</b></i> | <li><i><b>pR6K</b></i> | ||
| Line 151: | Line 151: | ||
<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> 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> | <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> | ||
| + | <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> was repeated with the new <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#DNAPurificationbyCentrifugation" target="_blank">purified</a> <i>pR6K</i>-cassette and <i>oprF</i> and different primer usage</li> | ||
| + | <ul> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> was initially performed 15 cycles without primer and afterwards 30 cycles with addition of primer</li> | ||
| + | </ul> | ||
| + | |||
</ul> | </ul> | ||
| + | </ul> | ||
| + | </ul> | ||
| + | <ul> | ||
| + | <li><b> RedET plasmid </b></li> | ||
| + | <ul> | ||
| + | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#Transformationviaelectroporation" target="_blank">Transformation</a> with electrocompotetent cells of the RedET plasmid into <i> E. coli KRX</i> | ||
| + | </ul> | ||
| + | |||
| + | |||
| + | |||
| + | </ul> | ||
</ul> | </ul> | ||
Revision as of 10:12, 1 October 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
- 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. Additionally we transform the RedET plasmid into the E. coli strain KRX.
- 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
- PCR amplification to connect the pR6K-cassette and oprF was repeated with the new purified pR6K-cassette and oprF and different primer usage
- PCR amplification was initially performed 15 cycles without primer and afterwards 30 cycles with addition of primer
- RedET plasmid
- Transformation with electrocompotetent cells of the RedET plasmid into E. coli KRX
- pR6K
