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Team:Bielefeld-CeBiTec/Notebook/Journal/Isobutanol/Jul
From 2014.igem.org
(Difference between revisions)
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<li><b>Backbone of pSB1C3</b></li> | <li><b>Backbone of pSB1C3</b></li> | ||
<ul> | <ul> | ||
| - | <li>We aim to amplify the backbone with Q5 polymerase</li> | + | <li>We aim to amplify the backbone with <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/KitsAndEnzymes#Polymerases" target="_blank">Q5 polymerase</a></li> |
<ul> | <ul> | ||
<li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_kivD_pSB1C3" target="_blank">fw_kivD_pSB1C3</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_alsS_pSB1C3" target="_blank">rv_alsS_pSB1C3</a>)</li> | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#PCR" target="_blank">PCR amplification</a> (<a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#fw_kivD_pSB1C3" target="_blank">fw_kivD_pSB1C3</a>, <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Primer#rv_alsS_pSB1C3" target="_blank">rv_alsS_pSB1C3</a>)</li> | ||
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<li>This week we aim to prove if the constructs were right</li> | <li>This week we aim to prove if the constructs were right</li> | ||
<ul> | <ul> | ||
| - | <li><a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Protocols#RestrictionDigestion" target="_blank">Restriction digestion</a> with <i>DpnI</i> | + | <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>DpnI</i></a></li> |
<ul> | <ul> | ||
<li>Bands (not) as expected (... bp)</li> | <li>Bands (not) as expected (... bp)</li> | ||
Revision as of 12:53, 27 August 2014
 |
July |
 |
- kivD, alsS, ilvC, ilvD and backbone of pSB1C3
- We tried to redo the amplification of last week.
- Optimization of PCR conditions for coding sequence amplification
- combinations of primers and templates as described before
- annealing temperature gradients from 50°C to 58°C were tried
- product amount was increased by lower annealing temperatures
- kivD, alsS, ilvC, ilvD and backbone of pSB1C3
- We tried to redo the amplification of last week.
- Optimization of PCR conditions for coding sequence amplification
- combinations of primers and templates as described before
- annealing temperature gradients from 50°C to 58°C were tried
- product amount was increased by lower annealing temperatures
- 54°C was identified as optimal annealing temperature
- 90 seconds were identified as optimal elongation time
- kivD, alsS, ilvC, ilvD
- With optimized conditions the amplification should give results this week
- PCR amplification as described before
- PCR products were extracted out of the gel.
- Plasmid purfication of the constructs
- Backbone of pSB1C3
- We aim to amplify the backbone with Q5 polymerase
- PCR amplification (fw_kivD_pSB1C3, rv_alsS_pSB1C3)
- Elongation time: ...
- Bands (not) as expected (... bp)
- PCR purification of backbone
- kivD, alsS, ilvC, ilvD
- This week we aim to prove if the constructs were right
- Restriction digestion with DpnI
- Bands (not) as expected (... bp)
- Gibson Assembly with kivD, alsS, ilvC and ilvD on pSB1C3
- Transformation with electrocompotetent cells
