Paris/July 30

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Contents

Sequences

All the biobricks that we got from the plate and all the ligations that succeeded up to now.


Colonies PCR screen

On colony PCRs performed to screen for correct ligation reactions (performed on 27/07/07):

10 clones of L1 ligation reactions, 6 clones of L6 ligation reactions

preparation of the following PCR mix

  • 18x (12.5µL 2xreaction mix)= 225
  • 18x (0.5µL oligo O18 VF2 10µM)=9
  • 18x (0.5µL oligo O19 VR 10µM)=9
  • 12µL H20+colony (10min 100°)

PCRs

PCR : P5 lox66-DapAColi
PCR Settings Buffer (5x) 5x 10µL Expected size
Annealing (°C) MgCl2 10µM 10µM 0µL 985
55 dNTP 10µM 10µM 1µL Success
Time Elongation Oligo F 10µM O6 2.5µL
2m00' Oligo R 10µM O7 2.5µL Image (click to enlarge)
Number cycles Water 34µl [[Image:|30px]]
30 Polymerase Phusion 0.5µL Band (0=ladder)
DNA Toothpick in MG16655 Glycerol


PCR : P6 Lox66-DapASubtilis
PCR Settings Buffer (5x) 5x 10µL Expected size
Annealing (°C) MgCl2 10µM 10µM 0µL 979
55 dNTP 10µM 10µM 1µL Success
Time Elongation Oligo F 10µM O8 2.5µL
2m00' Oligo R 10µM O9 2.5µL Image (click to enlarge)
Number cycles Water 34µl [[Image:|30px]]
30 Polymerase Phusion 0.5µL Band (0=ladder)
DNA 0.5µl Subtilis DNA


PCR : P7 Lox71-FtsZ
PCR Settings Buffer (5x) 5x 10µL Expected size
Annealing (°C) MgCl2 10µM 10µM 0µL 1260
55 dNTP 10µM 10µM 1µL Success
Time Elongation Oligo F 10µM O1 2.5µL
2m00' Oligo R 10µM O2 2.5µL Image (click to enlarge)
Number cycles Water 34µl [[Image:|30px]]
30 Polymerase Phusion 0.5µL Band (0=ladder)
DNA Toothpick in MG16655 Glycerol


PCR : P8 RBS-DapAColi
PCR Settings Buffer (5x) 5x 10µL Expected size
Annealing (°C) MgCl2 10µM 10µM 0µL 952
55 dNTP 10µM 10µM 1µL Success
Time Elongation Oligo F 10µM O20 2.5µL
2m00' Oligo R 10µM O7 2.5µL Image (click to enlarge)
Number cycles Water 34µl [[Image:|30px]]
30 Polymerase Phusion 0.5µL Band (0=ladder)
DNA Toothpick in MG16655 Glycerol


PCR : P11 Lox71-KmR-Lox66
PCR Settings Buffer (5x) 5x 10µL Expected size
Annealing (°C) MgCl2 10µM 10µM 0µL 1084
55 dNTP 10µM 10µM 1µL Success
Time Elongation Oligo F 10µM O10 2.5µL
2m00' Oligo R 10µM O11 2.5µL Image (click to enlarge)
Number cycles Water 34µl [[Image:|30px]]
30 Polymerase Phusion 0.5µL Band (0=ladder)
DNA 0.5 µl MP12

Digestion reactions

The following digestion reactions have been performed:

Digestion Products
Number Product Name Matrix Name Enzyme 1 Enzyme 2 size Description
D19' BB dig. lox66DapAsubtilis lox66-DapAsubtilis P6' PCR product EcoRI PstI ~960 PCR product digested for cloning as a BioBrick
D21' bw-insert lox66-DapAsubtilis lox66-DapAsubtilis P6' PCR product XbaI PstI ~960 PCR product digested for cloning as a BI
D27' lox71-ftsZ bw-insert lox71-ftsZ PCR product P7 XbaI PstI lox71-ftsZ(unmutated) ready for use as a backward insert
D33 RBS(B0030)CreORF L6' (clone 1 & 2) XbaI PstI RBS-Cre digestion for use as a BI
D34 lox66 L9' lox66 SpeI PstI Biobrick plasmid carrying lox66 digested as a BV
D35 lox71 L10' lox71 SpeI PstI Biobrick plasmid carrying lox71 digested as a BV

Ligations

Merci Eimad

MiniPreps

  • L1.1, L1.2
  • L6.1, L6.2