Team:Grenoble/Biology/Notebook/August/week 32

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August

Week 31 • Week 32 • Week 33 • Week 34 • Week 35

Week 32: August 06th to 12th

Goal of the week:

Assembly of pAra/Bad_RBS_GFP and RBS_Cya.

Monday, August 06th:

We wanted to check if the Gibson Assemblies (12/08/01) worked well.

To separate (protocol) the GA miniprep (12/08/03) 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.

photo_gel_27

Migration result for a 1.8% TAE agarose gel
(the DNA ladder scale is in kb)

  • Lane 1: pLAC_rsmY (pSB1A3) 3 miniprep product
  • Lane 2: pLAC_rsmY (pSB1A3) 2 miniprep product
  • Lane 3: pLAC_rsmY (pSB1A3) 1 miniprep product
  • Lane 4: pLAC_fha1_eCFP (pSB4C5) 2 miniprep product
  • Lane 5: pLAC_fha1_eCFP (pSB4C5) 1 miniprep product
  • Lane 6: DNA ladder 1kb (biolabs)
  • Lane 7: pAra/Bad_RBS_GFP_RBS_Cya (pSB3C5) 1 miniprep product
  • Lane 8: pAra/Bad_RBS_GFP_RBS_Cya (pSB3C5) 2 miniprep product
  • Lane 9: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 4 miniprep product
  • Lane 10: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 3 miniprep product
  • Lane 11: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 1 miniprep product
  • Lane 12: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 2 miniprep product
  • Lane 13: DNA ladder 1kb (biolabs)


We did some digestions (protocol) on GA miniprep (12/08/03). The digestions were achieved with XbaI during 10 minutes.

To separate (protocol) the digestion products, we prepared two 1.8% TAE agarose gels.
Migration conditions = 100V during 30 min.
In order to reveal the DNA fragments, we used EtBr.

photo_gel_28

Migration result for a 1.8% TAE agarose gel
(the DNA ladder scale is in kb)

  • Lane 1: data not shown pAra/Bad_RBS_GFP_RBS_Cya (pSB3C5) 1 digestion product
  • Lane 2: data not shown pAra/Bad_RBS_GFP_RBS_Cya (pSB3C5) 1 miniprep product
  • Lane 3: pAra/Bad_RBS_GFP_RBS_Cya (pSB3C5) 2 digestion product
  • Lane 4: pAra/Bad_RBS_GFP_RBS_Cya (pSB3C5) 2 miniprep product
  • Lane 5: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 4 digestion product
  • Lane 6: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 4 miniprep product
  • Lane 7: DNA ladder 1kb (biolabs)
  • Lane 8: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 3 digestion product
  • Lane 9: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 3 miniprep product
  • Lane 10: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 1 digestion product
  • Lane 11: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 1 miniprep product
  • Lane 12: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 2 digestion product
  • Lane 13: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 2 miniprep product

photo_gel_29

Migration result for a 1.8% TAE agarose gel
(the DNA ladder scale is in kb)

  • Lane 1: DNA ladder 1kb (biolabs)
  • Lane 2: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 4 digestion product
  • Lane 3: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 4 miniprep product
  • Lane 4: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 1 digestion product
  • Lane 5: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 1 miniprep product
  • Lane 6: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 2 digestion product
  • Lane 7: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 2 miniprep product
  • Lane 8: DNA ladder 80pb-10kb (fermentas)

The digestions showed no significant results.

Thursday, August 07th:

We did some verification PCRs on miniprep with HF Phusion enzyme (protocol) in order to check the constructions.

To separate (protocol) the PCR 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 PCR showed no significant results (data not shown).

Wednesday, August 08th:

We did some digestions (protocol) in order to check the construction: pAra/Bad_RBS_GFP_RBS_Cya and to do the construcion: pompC_mcherry.

We did an overlapping PCR on miniprep with HF Phusion enzyme (protocol) in order to do the construction: pAra/Bad_RBS_GFP_RBS_Cya.

To separate (protocol) the PCR products and the digestion products, we prepared 1.8% TAE agarose gels.
Migration conditions = 100V during 30 min.
In order to reveal the DNA fragments, we used EtBr.

The digestions experiments worked well, the PCR didn't work (data not shown).

Tuesday, August 09th:

We did some colony PCRs with HF Phusion enzyme (protocol) in order amplify RBS_Cya and pAra/Bad_RBS_GFP.

We realised a Gibson Assembly (protocol) to build: pSB3C5 with pAra/Bad_RBS_GFP and RBS_Cya.
With our Gibson Assembly products, we transformed (new protocol) BW25113 Cya- competent cells (protocol).

We did an overlapping PCR on miniprep with HF Phusion enzyme (protocol) in order to do the construction: pAra/Bad_RBS_GFP_RBS_Cya.
It showed no significant result (data not shown).

Friday, August 10th:

We relaunched in fresh LB the cultures of transformed cells made the day before (12/08/09).

We did a miniprep (protocol) on these transformed strains.

Conclusion of the week:

We didn't achieved to construct: pAra/Bad_RBS_GFP_RBS_Cya.

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