Paris/July 30

From 2007.igem.org

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== Ligations ==
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Merci Eimad

Revision as of 09:09, 31 July 2007

Contents

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