Paris/October 20

From 2007.igem.org

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== Ligation Reactions ==
== Ligation Reactions ==
 +
 +
{|
 +
|- style="background: #ccccff;"
 +
! colspan="5" style="background: #ccccff;" | Ligations
 +
|- style="background: #ccccff; text-align: center;"
 +
|width=5%| Number
 +
|width=20%| Insert
 +
|width=5%| Insert Volume (µL)
 +
|width=25%| Vector
 +
|width=5%| Vector Volume (µL)
 +
|width=35%| Comments
 +
|width=5%| Number of colonies
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |C22
 +
|
 +
|
 +
|D47
 +
|3
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |C23
 +
|
 +
|
 +
|D72
 +
|3
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |C24
 +
|
 +
|
 +
|D72 (from Oct. 14)
 +
|3
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |C25
 +
|
 +
|
 +
|D50
 +
|3
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |C26
 +
|
 +
|
 +
|D41
 +
|3
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |C27
 +
|
 +
|
 +
|D22
 +
|5
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |C28
 +
|
 +
|
 +
|D81
 +
|11
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |C29
 +
|
 +
|
 +
|D89
 +
|3
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |C30
 +
|
 +
|
 +
|D90
 +
|3
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L68
 +
|D56 - araC/pBad>>RBS-Cre BBinsert
 +
|10
 +
|D72 - pSB2K3 BBvector
 +
|3
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L68 bis
 +
|D56 (14/10/07) - araC/pBad>>RBS-Cre BBinsert
 +
|10
 +
|D72 (14/10/07) - pSB2K3 BBvector
 +
|3
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L69
 +
|D92
 +
|10
 +
|D50
 +
|3
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L70
 +
|D92
 +
|10
 +
|D41
 +
|3
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L71
 +
|D93
 +
|15
 +
|D50
 +
|3
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L72
 +
|D93
 +
|15
 +
|D41
 +
|3
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L73
 +
|D84
 +
|10
 +
|D22
 +
|5
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L74
 +
|D80
 +
|3
 +
|D81
 +
|15
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L74 bis
 +
|D80
 +
|4
 +
|D81
 +
|12
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L75
 +
|D83
 +
|4
 +
|D89
 +
|3
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L76
 +
|D83
 +
|10
 +
|D90
 +
|3
 +
|
 +
|0
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |L77
 +
|D94.6
 +
|3
 +
|D95
 +
|10
 +
|
 +
|0
 +
|}
== Culture ==
== 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.
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.

Revision as of 16:16, 23 October 2007

yesterday -- tomorrow

Contents

Digestion Reaction

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.

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