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Team:Grenoble/Biology/Notebook/July/week 29
From 2012.igem.org
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In order to reveal the DNA fragments, we used EtBr.<br/> | In order to reveal the DNA fragments, we used EtBr.<br/> | ||
<br/> | <br/> | ||
| - | <center><img src="https://static.igem.org/mediawiki/2012/ | + | <center><img src="https://static.igem.org/mediawiki/2012/3/33/120717.jpg" alt="photo_gel_11"/></center> |
| - | + | <u>Migration result for a 1.8% TAE agarose gel</u><br/> | |
| + | <i>(the DNA ladder scale is in kb)</i><br/> | ||
| + | Lane 1: DNA ladder 1kb (biolabs)<br/> | ||
| + | Lane 2-3: the deposits have failed<br/> | ||
| + | Lane 4: pSB4K5 colony PCR product (DMSO)<br/> | ||
| + | Lane 5: pSB4K5 colony PCR product<br/> | ||
| + | Lane 6: pSB4K5 PCR (miniprep) product (DMSO)<br/> | ||
| + | Lane 7: pSB4K5 PCR (miniprep) product<br/> | ||
| + | Lane 8: DNA ladder 1kb (biolabs)<br/> | ||
| + | Lane 9: DNA ladder 100bp (biolabs)<br/> | ||
| + | Lane 10: pLAC (fha1) PCR product (DMSO)<br/> | ||
| + | Lane 11: pLAC (fha1) PCR product<br/> | ||
| + | Lane 12: pLAC_RBS PCR product (DMSO)<br/> | ||
| + | Lane 13: pLAC_RBS PCR product<br/> | ||
| + | Lane 14: pLAC (rsmY) PCR product (DMSO)<br/> | ||
| + | Lane 15: pLAC (rsmY) PCR product<br/> | ||
| + | Lane 16: fha1 PCR product (DMSO)<br/> | ||
| + | Lane 17: fha1 PCR product<br/> | ||
| + | Lane 18: empty. | ||
| + | Lane 19: pSB4K5 miniprep product | ||
| + | Lane 20: plasmid with pAra/Bad_RBS_GFP miniprep product | ||
</section> | </section> | ||
</body> | </body> | ||
Revision as of 06:05, 7 August 2012
Week 29: July 16th to July 22nd
Goal of the week
test the Gibson Assembly and the transformation protocols, recover and amplify some biobricks involved in our genetic networks :- pAra/Bad_RBS_GFP (1300bp)
- RBS_Cya (2600bp)
- fha1 (80bp)
- eCFP (800bp)
- pSB4K5 (2400bp)
Monday, July 16th:
Precultured cells are prepared:- Strains = BW25113 WT and BW25113 cya- pAra/Bad
- Conditions = Conditions = LB liquid medium, 37°C, 200rpm, overnight
Tuesday, July 17th:
Using iGEM 2012 biobricks and the Gibson Assembly product we transformed (protocol) BW25113 WT cells. We obtained four transformed strains with:- BBa_I13601: pLAC_RBS
- BBa_E0422: eCFP
- 120713PP_GA_001 (rsmY)
- 120713PP_GA_002 (RsmA)
We did a miniprep (protocol kit: Nucleospin plasmid Quick Pure) on 2 strains, on which we wanted to recover pSB4K5 and the plasmid with pAra/Bad_RBS_GFP:
- transformed strain with pSB4K5
- BW25113 cya- pAra/Bad
We did PCRs with HF Phusion enzyme (protocol) on this miniprep in order to amplify pSB4K5, and from colony to amplify pLAC (fha), pLAC (rsmY), pLAC_RBS, fha and pSB4K5. We did the PCR both with and without DMSO.
To separate the PCR products and the miniprep products, we prepared a 1.8% TAE agarose gel.
Migration conditions = 100V during 30 min.
In order to reveal the DNA fragments, we used EtBr.
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 1kb (biolabs)
Lane 2-3: the deposits have failed
Lane 4: pSB4K5 colony PCR product (DMSO)
Lane 5: pSB4K5 colony PCR product
Lane 6: pSB4K5 PCR (miniprep) product (DMSO)
Lane 7: pSB4K5 PCR (miniprep) product
Lane 8: DNA ladder 1kb (biolabs)
Lane 9: DNA ladder 100bp (biolabs)
Lane 10: pLAC (fha1) PCR product (DMSO)
Lane 11: pLAC (fha1) PCR product
Lane 12: pLAC_RBS PCR product (DMSO)
Lane 13: pLAC_RBS PCR product
Lane 14: pLAC (rsmY) PCR product (DMSO)
Lane 15: pLAC (rsmY) PCR product
Lane 16: fha1 PCR product (DMSO)
Lane 17: fha1 PCR product
Lane 18: empty. Lane 19: pSB4K5 miniprep product Lane 20: plasmid with pAra/Bad_RBS_GFP miniprep product
