Paris/October 20

From 2007.igem.org

(Difference between revisions)
(→PCR reaction)
(→PCR reaction)
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== PCR reaction ==
== PCR reaction ==
 +
 +
{{Paris_PCR_0| Title = PrefixBB-frt1-CmR-frt2-SufixBB
 +
|Denaturation= 98°C 2' first / 20" then
 +
|Annealing= /
 +
|Elongation= 72°C 1'40"
 +
|Cycles= 30
 +
|Final elongation= 72°C 10'
 +
|Buffer=  (5x) 10µL
 +
|MgCl2= 10µM 0µL (no extra)
 +
|dNTP= 10µM 1µL
 +
|n_oligoF= O54
 +
|v_oligoF= 10µM 4µL
 +
|n_oligoR= O55
 +
|v_oligoR= 10µM 4µL
 +
|water= qsp 50µL
 +
|pol= Phusion 0,5µL
 +
|DNA= pY-2P-IntC 5µL
 +
|Size= 1000bp
 +
|Success=YES
 +
|Image=
 +
|Band=
 +
|}}
 +
 +
{{Paris_PCR_0| Title = PrefixBB-frt1-CmR-frt2-SufixBB
 +
|Denaturation= 98°C 2' first / 20" then
 +
|Annealing= /
 +
|Elongation= 72°C 1'40"
 +
|Cycles= 30
 +
|Final elongation= 72°C 10'
 +
|Buffer=  (5x) 10µL  ! GC one !
 +
|MgCl2= 10µM 0µL (no extra)
 +
|dNTP= 10µM 1µL
 +
|n_oligoF= O54
 +
|v_oligoF= 10µM 4µL
 +
|n_oligoR= O55
 +
|v_oligoR= 10µM 4µL
 +
|water= qsp 50µL
 +
|pol= Phusion 0,5µL
 +
|DNA= pY-2P-IntC 5µL
 +
|Size= 1000bp
 +
|Success=NO
 +
|Image=
 +
|Band=
 +
|}}
 +
 +
{{Paris_PCR_0| Title = PrefixBB-frt1-CmR-frt2-SufixBB
 +
|Denaturation= 98°C 2' first / 20" then
 +
|Annealing= /
 +
|Elongation= 72°C 1'40"
 +
|Cycles= 30
 +
|Final elongation= 72°C 10'
 +
|Buffer=  (5x) 10µL ! GC one !
 +
|MgCl2= 10µM 0µL (no extra)
 +
|DMSO= 2,5µL (or 5%)
 +
|dNTP= 10µM 1µL
 +
|n_oligoF= O54
 +
|v_oligoF= 10µM 4µL
 +
|n_oligoR= O55
 +
|v_oligoR= 10µM 4µL
 +
|water= qsp 50µL
 +
|pol= Phusion 0,5µL
 +
|DNA= pY-2P-IntC 5µL
 +
|Size= 1000bp
 +
|Success=NO
 +
|Image=
 +
|Band=
 +
|}}
 +
 +
{{Paris_PCR_0| Title = PrefixBB-frt1-CmR-frt2-T1T2-ufixBB
 +
|Denaturation= 98°C 2' first / 20" then
 +
|Annealing= /
 +
|Elongation= 72°C 1'40"
 +
|Cycles= 30
 +
|Final elongation= 72°C 10'
 +
|Buffer=  (5x) 10µL
 +
|MgCl2= 10µM 0µL (no extra)
 +
|dNTP= 10µM 1µL
 +
|n_oligoF= O54
 +
|v_oligoF= 10µM 4µL
 +
|n_oligoR= O56
 +
|v_oligoR= 10µM 4µL
 +
|water= qsp 50µL
 +
|pol= Phusion 0,5µL
 +
|DNA= pY-2P-IntC 5µL
 +
|Size= 1000bp
 +
|Success=YES
 +
|Image=
 +
|Band=
 +
|}}
{{Paris_PCR_0| Title = PrefixBB-frt1-CmR-frt2-T1T2-SufixBB
{{Paris_PCR_0| Title = PrefixBB-frt1-CmR-frt2-T1T2-SufixBB
-
|Annealing= 60
+
|Denaturation= 98°C 2' first / 20" then
-
|Elongation= 2'30
+
|Annealing= /
-
|Cycles= 33
+
|Elongation= 72°C 1'40"
-
|Buffer=  5x 10µL (GC rich)
+
|Cycles= 30
 +
|Final elongation= 72°C 10'
 +
|Buffer=  (5x) 10µL ! GC one !
|MgCl2= 10µM 0µL (no extra)
|MgCl2= 10µM 0µL (no extra)
|dNTP= 10µM 1µL
|dNTP= 10µM 1µL
-
|n_oligoF= O51
+
|n_oligoF= O54
-
|v_oligoF= 10µM 2.5µL
+
|v_oligoF= 10µM 4µL
-
|n_oligoR= O53
+
|n_oligoR= O56
-
|v_oligoR= 10µM 2.5µL
+
|v_oligoR= 10µM 4µL
|water= qsp 50µL
|water= qsp 50µL
|pol= Phusion 0,5µL
|pol= Phusion 0,5µL
-
|DNA= pY-2P-IntC 1µL (dil. 1/10)
+
|DNA= pY-2P-IntC 5µL
-
|Size=  
+
|Size= 1000bp
|Success=NO
|Success=NO
 +
|Image=
 +
|Band=
 +
|}}
 +
 +
{{Paris_PCR_0| Title = PrefixBB-frt1-CmR-frt2-T1T2-SufixBB
 +
|Denaturation= 98°C 2' first / 20" then
 +
|Annealing= /
 +
|Elongation= 72°C 1'40"
 +
|Cycles= 30
 +
|Final elongation= 72°C 10'
 +
|Buffer=  (5x) 10µL ! GC one !
 +
|MgCl2= 10µM 0µL (no extra)
 +
|DMSO= 2,5µL (or 5%)
 +
|dNTP= 10µM 1µL
 +
|n_oligoF= O54
 +
|v_oligoF= 10µM 4µL
 +
|n_oligoR= O56
 +
|v_oligoR= 10µM 4µL
 +
|water= qsp 50µL
 +
|pol= Phusion 0,5µL
 +
|DNA= pY-2P-IntC 5µL
 +
|Size= 1000bp
 +
|Success=YES
|Image=
|Image=
|Band=
|Band=

Revision as of 14:39, 22 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

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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