Team:Grenoble/Biology/Notebook/August
From 2012.igem.org
Revision as of 11:35, 13 August 2012 by Adeline millet (Talk | contribs)
August
week 31 week 32 week 33 week 34 week 35Week 31: July 30th to August 05th
Goal of the week:
We wanted to recover and amplify some biobricks involved in our genetic networks :- pSB4C5 (2400bp)
- pompC (100bp)
- mcherry (900bp)
- eCFP (800bp)
- GFP (100bp)
Tuesday, July 31th:
We did a miniprep (protocol kit: Nucleospin plasmid Quick Pure) on the transformed strain with pSB4C5 (12/07/25), on which we wanted to recover pSB4C5.We did some colony PCRs (on 12/07/25 colony) and PCRs on miniprep with HF Phusion enzyme (protocol) in order to amplify pSB4C5.
Annealing temperature = 60°C.
To separate 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.
Migration result for a 1.8% TAE agarose gel
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 1kb (biolabs)
Lane 2: pSB4C5 (miniprep) PCR product (DMSO)
Lane 3: pSB4C5 (miniprep) PCR product (DMSO)
Lane 4: pSB4C5 (miniprep) PCR product
Lane 5: pSB4C5 (miniprep) PCR product
Lane 6: pSB4C5 (colony) PCR product (DMSO)
Lane 7: pSB4C5 (colony) PCR product (DMSO)
Lane 8: pSB4C5 (colony) PCR product
Lane 9: pSB4C5 (colony) PCR product
Lane 10: DNA ladder 1kb (biolabs)
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 1kb (biolabs)
Lane 2: pSB4C5 (miniprep) PCR product (DMSO)
Lane 3: pSB4C5 (miniprep) PCR product (DMSO)
Lane 4: pSB4C5 (miniprep) PCR product
Lane 5: pSB4C5 (miniprep) PCR product
Lane 6: pSB4C5 (colony) PCR product (DMSO)
Lane 7: pSB4C5 (colony) PCR product (DMSO)
Lane 8: pSB4C5 (colony) PCR product
Lane 9: pSB4C5 (colony) PCR product
Lane 10: DNA ladder 1kb (biolabs)
We only saw primer dimer bands, there was a PCR condition problem.
We did some PCRs on miniprep with HF Phusion enzyme (protocol) in order to amplify pSB4C5.
Annealing temperature = 65°C.
We did some digestions (protocol) on miniprep in order to do the construction: pAra/Bad_RBS_GFP_RBS_Cya. The digestions were achieved with two restriction enzymes : XbaI and SpeI during 10 minutes.
To separate the PCR products and the digestion 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.
Migration result for a 1.8% TAE agarose gel
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 1kb (biolabs)
Lane 2: pSBAC5 digestion product
Lane 3: pSB4C5 digestion product
Lane 4: pSB4C5 (miniprep) PCR product (DMSO)
Lane 5: pSB4C5 (miniprep) PCR product (DMSO)
Lane 6: pSB4C5 (miniprep) PCR product
Lane 7: pSB4C5 (miniprep) PCR product
Lane 8: pSB4C5 (colony) PCR product (DMSO)
Lane 9: pSB4C5 (colony) PCR product (DMSO)
Lane 10: pSB4C5 (colony) PCR product
Lane 11: pSB4C5 (colony) PCR product
Lane 12: DNA ladder 1kb (biolabs)
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 1kb (biolabs)
Lane 2: pSBAC5 digestion product
Lane 3: pSB4C5 digestion product
Lane 4: pSB4C5 (miniprep) PCR product (DMSO)
Lane 5: pSB4C5 (miniprep) PCR product (DMSO)
Lane 6: pSB4C5 (miniprep) PCR product
Lane 7: pSB4C5 (miniprep) PCR product
Lane 8: pSB4C5 (colony) PCR product (DMSO)
Lane 9: pSB4C5 (colony) PCR product (DMSO)
Lane 10: pSB4C5 (colony) PCR product
Lane 11: pSB4C5 (colony) PCR product
Lane 12: DNA ladder 1kb (biolabs)
For the PCR results, we only saw primer dimer bands, there was a PCR condition problem.
The digestion worked well, we realised a DNA extraction (protocol kit: Nucleospin extract II) from the two digestion products (code = 120731AM_DIG_024).
Wednesday, August 01st:
We did a touchdown PCR (from 70°C to 60°C) from miniprep with HF Phusion enzyme (protocol) in order to amplify pSB4C5.To separate the PCR products and the digestion 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.
Migration result for a 1.8% TAE agarose gel
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 1kb (biolabs)
Lane 2: pSBAC5 (digestion) PCR product
Lane 3: pSB4C5 (digestion) PCR product (DMSO)
Lane 4: pSB4C5 (miniprep) PCR product
Lane 5: pSB4C5 (miniprep) PCR product (DMSO)
Lane 6: DNA ladder 1kb (biolabs)
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 1kb (biolabs)
Lane 2: pSBAC5 (digestion) PCR product
Lane 3: pSB4C5 (digestion) PCR product (DMSO)
Lane 4: pSB4C5 (miniprep) PCR product
Lane 5: pSB4C5 (miniprep) PCR product (DMSO)
Lane 6: DNA ladder 1kb (biolabs)
We only saw primer dimer bands, there was a PCR condition problem.
We realised four Gibson Assemblies (protocol) to build four plasmids:
- pSB4C5 (fha1) (120726AM_PCR_021) with pLAC (fha1) (120713PP_PCR_009), fha1 (120723AM_PCR_020) and eCFP (120720_DIG_019)
=> 120801PP_GA_003 - pSB1A3 (120713PP_PCR_014) with pLAC (rsmY) (120713PP_PCR_011) and rsmY (120713PP_PCR_013)
=> 120801PP_GA_004 - pSB3C5 (Cya) (120713PP_PCR_015) with pAra/Bad_RBS_GFP (120727AM_PCR_023) and RBS_Cya (120727AM_PCR_022)
=> 120801PP_GA_005 - pSB4C5 (120731AM_DIG_024) with pAra/Bad_RBS_GFP (120727AM_PCR_023) and RBS_Cya (120727AM_PCR_022)
=> 120713PP_GA_006