Paris/October 20

From 2007.igem.org

yesterday -- tomorrow

Contents

Digestion Reaction

Digestion Products
Number Product Name Matrix Name Enzyme 1 Enzyme 2 Size Description
D79 pJ23100>>lox71-B0015(T) L44.1 Xba1 Pst1 BI
D80 pJ23100>>lox71-B0015(T) L44.2 Xba1 Pst1 BI
D81 pJ23107>>lox71-B0015(T) L45.1 Xba1 Pst1 BI
D82 pJ23107>>lox71-B0015(T) L45.2 Xba1 Pst1 BI
D83 pTet>>lox71-B0015(T) L46.1 Xba1 Pst1 BI
D84 araC/pBad>>lox71-B0015(T) L47.1 Xba1 Pst1 BI
D85 B0015(T)-lox66-RBSDapAcoli L53.7 EcoRI SpeI FI
D86 attB-lox66-RBSDapAcoli L54.3 EcoRI SpeI FI
D87 attB-lox66-RBSDapAcoli L54.4 EcoRI SpeI FI
D88 B0015(T)-lox66-RBSdapAsubtilis L56.3 EcoRI SpeI FI
D89 attB-lox66-RBSdapAsubtilis L57.1 EcoRI SpeI FI
D90 attB-lox66-RBSdapAsubtilis L57.3 EcoRI SpeI FI
D91 BBa_J61025 MP14.1 EcoRI XbaI FV - Medium constitutive promoter in J61002 (cl. 1)
D92 BBa_J61025 MP14.2 EcoRI XbaI FV - Medium constitutive promoter in J61002 (cl. 2)
D93 BBa_J61025 MP14.1 Spe1 Pst1 BV - Medium constitutive promoter in J61002 (cl. 1)
D94 BBa_J61025 MP14.2 Spe1 Pst1 BV - Medium constitutive promoter in J61002 (cl. 2)

PCR reaction

PCR : PrefixBB-frt1-CmR-frt2-SufixBB
PCR Settings Buffer (5x) (5x) 10µL Expected size
Annealing (°C) MgCl2 10µM 10µM 0µL (no extra) 1000bp
/ dNTP 10µM 10µM 1µL Success
Time Elongation Oligo F 10µM O54 10µM 4µL YES
72°C 1'40" Oligo R 10µM O55 10µM 4µL Image (click to enlarge)
Number cycles Water qsp 50µL [[Image:|30px]]
30 Polymerase Phusion 0,5µL Band (0=ladder)
DNA pY-2P-IntC 5µL
PCR : PrefixBB-frt1-CmR-frt2-SufixBB
PCR Settings Buffer (5x) (5x) 10µL  ! GC one ! Expected size
Annealing (°C) MgCl2 10µM 10µM 0µL (no extra) 1000bp
/ dNTP 10µM 10µM 1µL Success
Time Elongation Oligo F 10µM O54 10µM 4µL NO
72°C 1'40" Oligo R 10µM O55 10µM 4µL Image (click to enlarge)
Number cycles Water qsp 50µL [[Image:|30px]]
30 Polymerase Phusion 0,5µL Band (0=ladder)
DNA pY-2P-IntC 5µL
PCR : PrefixBB-frt1-CmR-frt2-SufixBB
PCR Settings Buffer (5x) (5x) 10µL ! GC one ! Expected size
Annealing (°C) MgCl2 10µM 10µM 0µL (no extra) 1000bp
/ dNTP 10µM 10µM 1µL Success
Time Elongation Oligo F 10µM O54 10µM 4µL NO
72°C 1'40" Oligo R 10µM O55 10µM 4µL Image (click to enlarge)
Number cycles Water qsp 50µL [[Image:|30px]]
30 Polymerase Phusion 0,5µL Band (0=ladder)
DNA pY-2P-IntC 5µL
PCR : PrefixBB-frt1-CmR-frt2-T1T2-ufixBB
PCR Settings Buffer (5x) (5x) 10µL Expected size
Annealing (°C) MgCl2 10µM 10µM 0µL (no extra) 1000bp
/ dNTP 10µM 10µM 1µL Success
Time Elongation Oligo F 10µM O54 10µM 4µL YES
72°C 1'40" Oligo R 10µM O56 10µM 4µL Image (click to enlarge)
Number cycles Water qsp 50µL [[Image:|30px]]
30 Polymerase Phusion 0,5µL Band (0=ladder)
DNA pY-2P-IntC 5µL
PCR : PrefixBB-frt1-CmR-frt2-T1T2-SufixBB
PCR Settings Buffer (5x) (5x) 10µL  ! GC one ! Expected size
Annealing (°C) MgCl2 10µM 10µM 0µL (no extra) 1000bp
/ dNTP 10µM 10µM 1µL Success
Time Elongation Oligo F 10µM O54 10µM 4µL NO
72°C 1'40" Oligo R 10µM O56 10µM 4µL Image (click to enlarge)
Number cycles Water qsp 50µL [[Image:|30px]]
30 Polymerase Phusion 0,5µL Band (0=ladder)
DNA pY-2P-IntC 5µL
PCR : PrefixBB-frt1-CmR-frt2-T1T2-SufixBB
PCR Settings Buffer (5x) (5x) 10µL ! GC one ! Expected size
Annealing (°C) MgCl2 10µM 10µM 0µL (no extra) 1000bp
/ dNTP 10µM 10µM 1µL Success
Time Elongation Oligo F 10µM O54 10µM 4µL YES
72°C 1'40" Oligo R 10µM O56 10µM 4µL Image (click to enlarge)
Number cycles Water qsp 50µL [[Image:|30px]]
30 Polymerase Phusion 0,5µL Band (0=ladder)
DNA pY-2P-IntC 5µL

Purification of Digestion and PCR products

Ligation Reactions

Ligations
Number Insert Insert Volume (µL) Vector Vector Volume (µL) Comments Number of colonies
C22 D47 3 0
C23 D72 3 0
C24 D72 (from Oct. 14) 3 0
C25 D50 3 0
C26 D41 3 0
C27 D22 5 0
C28 D81 11 0
C29 D89 3 0
C30 D90 3 0
L68 D56 - araC/pBad>>RBS-Cre BBinsert 10 D72 - pSB2K3 BBvector 3 0
L68 bis D56 (14/10/07) - araC/pBad>>RBS-Cre BBinsert 10 D72 (14/10/07) - pSB2K3 BBvector 3 0
L69 D92 10 D50 3 0
L70 D92 10 D41 3 0
L71 D93 15 D50 3 0
L72 D93 15 D41 3 0
L73 D84 10 D22 5 0
L74 D80 3 D81 15 0
L74 bis D80 4 D81 12 0
L75 D83 4 D89 3 0
L76 D83 10 D90 3 0
L77 D94.6 3 D95 10 0

Culture

Launching of 80mL culture of the strain countaining the plasmid pY-2P-IntC, last minipreps revealed the weak nomber of plasmid recover with a classic miniprep on 5mL culture.