Team:Grenoble/Biology/Notebook/June/week 28

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Week 28: July 09th to July 15th

Goal of the week

we wanted to 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)
We also planned to realise the gibson assemblies for the first constructions.

Monday, July 09th:

Precultured cells are prepared:
  • Strains = BW25113 WT and BW25113 cya- pAra/Bad
  • Conditions = LB liquid medium, 37°C, 200rpm, overnight

Tuesday, July 10th:

For the PCRs, we changed the enzyme from GoTaq to High Fidelity (HF) Phusion.
We did some colony PCR with a HF Phusion 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.
We did the PCR both with and without DMSO (Amplification of difficult targets, such as those with GC-rich sequences or secondary structure, may be improved by the presence of additives such as DMSO).

To separate the PCR products, we prepared two gels:
  • a 1.3% 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 = 50V during 1h15.
We used EtBr to reveal the DNA fragments.
photo_gel_4
Migration result for the 1.3% TAE agarose gel (small fragments)
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 100bp (biolabs)
Lane 2 and 3: fha1 PCR product
Lane 4 and 5: RsmA PCR product
Lane 6 and 7: rsmY PCR product
Lane 8 and 9: fha1 PCR product (DMSO)
Lane 10 and 11: RsmA PCR product (DMSO)
Lane 12: rsmY PCR product (DMSO)
Lane 13: DNA ladder 100bp (biolabs)

photo_gel_5
Migration result for the 0.8% TAE agarose gel (big fragments)
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 1kb (biolabs)
Lane 2 and 3: RBS_Cya PCR product
Lane 4 and 5: pSB1A3 PCR product
Lane 6 and 7: pAra/Bad_RBS_GFP PCR product
Lane 8 and 9: RBS_Cya PCR product (DMSO)
Lane 10 and 11: pSBA13 PCR product (DMSO)
Lane 12: pAra/Bad_GFP PCR product (DMSO)
Lane 13: DNA ladder 1kb (biolabs)

There was a migration problem on the second gel (DNA ladder migration was not right) and we only saw primer dimer bands. There was a PCR condition problem.

Precultured cells were prepared:
  • Strains = BW25113 WT.
  • Conditions = LB liquid medium, 37°C, 200rpm, overnight.

Wednesday, July 11th:

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