Team:Grenoble/Biology/Notebook/July
From 2012.igem.org
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Lane 8 and 9: rsmY PCR product<br/> | Lane 8 and 9: rsmY PCR product<br/> | ||
Lane 10: DNA ladder 100bp (biolabs)<br/> | Lane 10: DNA ladder 100bp (biolabs)<br/> | ||
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<center><img src="https://static.igem.org/mediawiki/2012/6/67/120705.jpg" alt="photo_gel_2"/></center> | <center><img src="https://static.igem.org/mediawiki/2012/6/67/120705.jpg" alt="photo_gel_2"/></center> | ||
<u>Migration results for the 0.8% TAE agarose gel (big fragments)</u><br> | <u>Migration results for the 0.8% TAE agarose gel (big fragments)</u><br> |
Revision as of 14:10, 6 August 2012
Week 27: July 02nd to July 08st
Goal of the week
We wanted to test the PCR conditions, recover and amplify the biobricks involved in our genetic networks :- pAra/Bad_RBS_GFP (1300bp)
- RBS_Cya (2600bp)
- pLAC (100bp)
- fha (80bp)
- eCFP (800bp)
- pLAC_RBS (120bp)
- RsmA (200bp)
- rsmY (170bp)
- pSB1A3 (2400bp)
- pSB4K5 (2400bp)
- pSB3C5 (2400bp)
Tuesday, July 03rd:
Precultured cells are prepared:- Strains = BW25113 WT, BW25113 cya- and BW25113 cya- pAra/Bad
- Conditions = LB liquid medium, 37°C, 200rpm, overnight
Wednesday, July 04th:
We did PCRs on colony with a GoTaq enzyme (protocol) to amplify:- fha1, RsmA, rsmY and pSB1A3 from iGEM Grenoble 2011 glycerol stock.
- pAra/Bad_RBS_GFP from BW25113 cya- pAra/Bad precultured cells.
- RBS_Cya from BW25113 WT precultured cells.
Using iGEM 2012 biobricks we transformed BW25113 WT cells (protocol). We obtained four transformed strains with four different biobricks:
- BBa_I13601: pLAC_RBS
- BBa_E0422: eCFP
- pSB3C5 plasmid
- psB4K5 plasmid
Thursday, July 5th:
The transformations (12/07/04) show no growth on LB Agar plate + antibiotics.In order to separate the PCR products (12/07/04), we prepared two gels:
- a 1.3% Tris base Acetic acid EDTA (TAE) agarose gel, for the small fragments: RsmA, fha1 and rsmY (length < 1000bp)
- a 0.8% TAE agarose gel, for the big fragments: pSB1A3, RBS_Cya, and pAra/Bad_RBS_GFP (length > 1000bp)
Migration conditions = 100V during 35 min.
In order to reveal the fragments, we used Ethidium Bromide (EtBr).
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 100bp (biolabs)
Lane 2 and 3: not iGEM PCR products
Lane 4 and 5: RsmA PCR product
Lane 6 and 7: fha1 PCR product
Lane 8 and 9: rsmY PCR product
Lane 10: DNA ladder 100bp (biolabs)
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 1kb (biolabs)
Lane 2 and 3: not iGEM PCR products
Lane 4 and 5: pSB1A3 PCR product
Lane 6 and 7: RBS_Cya PCR product
Lane 8 and 9: pAra/Bad_RBS_GFP PCR product
Lane 10: DNA ladder 1kb (biolabs)
There is no visible migration on the second gel (big fragments). We realized a DNA extraction from the three small fragments (protocol kit: Nucleospin extract II):
- fha1 (PCR product code = 120705PP_PCR_001 and 120705PP_PCR_002)
- RsmA (PCR product code = 120705PP_PCR_003 and 120705PP_PCR_004)
- rsmY (PCR product code = 120705PP_PCR_005 and 120705PP_PCR_006)
Precultured cells are prepared:
- Strains = iGEM Grenoble 2011 glycerol stock with fha, RsmA and rsmY.
- Conditions = LB liquid medium, 37°C, 200rpm, overnight.
We did colony PCR with a GoTaq enzyme (protocol) to amplify:
- RBS_Cya from BW25113 WT strain
- pSB1A3 from iGEM 2011 glycerol stock
To separate the PCR products, we prepared a 0.8% TAE agarose gel.
Migration conditions = 50V during 1h35.
The DNA fragments were revealed with EtBr.