Paris/July 23

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Revision as of 23:07, 23 July 2007 by David.bikard (Talk | contribs)

Contents

w121 growth

w121 culture launched on 22/07/07 did not grow ON: I had forgot to add DAP to LB-Erythro growth medium.

at 12h30, the following culture was launched:

  • w121 in 2ml LB-Erythro-DAP

We were unable to perform growth kinetics analysis of w121 in different DAP-supplemented media (because the machine is occupied for the night

Transduction of DapA deletion in MG1655

  • The transduction seems to have worked: isolation of clones on LB and LB+DAP for verification.

Migration of digestion products

MiniPrep


Digestion reactions

Each of the digestion reactions that follow were performed as follows:

  • 20µL DNA solution (miniprep or PCR product)
  • 5µL Buffer 10x
  • 0.5µL BSA 100x
  • 2µL of each of the 2 enzymes indicated
  • H20 qsp 50µL

NEB3 buffer used for XbaI/PstI double digestion NEBEcoRI buffer used for both EcoRI/SpeI & EcoRI/PstI double digestion reactions


Digestion Products
Number Product Name Matrix Name Enzyme 1 Enzyme 2 size Description
D22 pSB1A2 open vector BBa_J61047 (MP9.1 & MP9.2) EcoI PstI Open pSB1A2 vector for BioBrick EcoRI/PstI insertio
D23 AraC/pBad promoter fw-insert BBa_I0500 (MP4.1 & MP4.2) EcoI SpeI AraC/pBad promoter ready for use as a forward insert
D24 lox71-ftsZ bw-insert lox71-ftsZ PCR product P7 XbaI PstI lox71-ftsZ(unmutated) ready for use as a backward insert
D25 eCFP bw-insert BBa_E0422 (MP6.1 & MP6.2) XbaI PstI eCFP (RBS-OFR_LVA-T) ready for use as a backward insert
D26 PoPs to GFP converter BBa_E0421 (MP7.1 & MP7.2) XbaI PstI PoPs to GFP converter ready for use as a backward insert
D27 gfp-tripart BBa_E0840 (MP8.1 & MP8.2) XbaI PstI gfp-tri part (strongRBS-ORF-T) ready for use as a backward insert

PCRs

PCR : Lox71-FtsA-FtsZ
PCR Settings Buffer (5x) 5x 10µL Expected size
Annealing (°C) MgCl2 10µM 10µM 0µL 2058
60 dNTP 10µM 10µM 1µL Success
Time Elongation Oligo F 10µM 3 Lox71-FtsA-F 2.5µL
3m00' Oligo R 10µM 2 FtsZ-R 2.5µL Image (click to enlarge)
Number cycles Water 34µl [[Image:|30px]]
35 Polymerase Phusion 0.5µL Band (0=ladder)
DNA Toothpick in MG16655 Glycerol


PCR : Assembly PCR Lox71-FtsA-FtsZ-1 + FtsZ-2
PCR Settings Buffer (5x) 5x 10µL Expected size
Annealing (°C) MgCl2 10µM 10µM 0µL 2058
70°C (5x) without oligos + 60°C (35x) dNTP 10µM 10µM 1µL Success
Time Elongation Oligo F 10µM 3 Lox71-FtsA-F 2.5µL
3m00' Oligo R 10µM 2 FtsZ-R 2.5µL Image (click to enlarge)
Number cycles Water 34µl [[Image:|30px]]
40x Polymerase Phusion 0.5µL Band (0=ladder)
DNA 8.5µl Lox71-FtsA-FtsZ-1 (~50ng) + 17.5µl FtsZ-2 (~50ng)


PCR : B0030-DapAColi
PCR Settings Buffer (5x) 5x 10µL Expected size
Annealing (°C) MgCl2 10µM 10µM 0µL 952
60°C dNTP 10µM 10µM 1µL Success
Time Elongation Oligo F 10µM o20 RBS-DapAColi 2.5µL
3m00' Oligo R 10µM o7 DapAColi-R 2.5µL Image (click to enlarge)
Number cycles Water 34µl [[Image:|30px]]
35x Polymerase Phusion 0.5µL Band (0=ladder)
DNA Toothpick in MG16655 Glycerol


PCR : B0030-DapASubtilis
PCR Settings Buffer (5x) 5x 10µL Expected size
Annealing (°C) MgCl2 10µM 10µM 0µL 946
60°C dNTP 10µM 10µM 1µL Success
Time Elongation Oligo F 10µM o20 RBS-DapASubtilis 2.5µL
3m00' Oligo R 10µM o9 DapASubtilis-R 2.5µL Image (click to enlarge)
Number cycles Water 34µl [[Image:|30px]]
35x Polymerase Phusion 0.5µL Band (0=ladder)
DNA Toothpick in MG16655 Glycerol