Team:Paris/July 17

From 2008.igem.org

(Difference between revisions)
(→Results)
(→Screening PCR of the transformations)
 
(31 intermediate revisions not shown)
Line 105: Line 105:
-
==Screening PCR of the transformations==
+
==PCR Screening of the transformants==
Use of the same 8 clones of Biobricks that have been tested for cloning  
Use of the same 8 clones of Biobricks that have been tested for cloning  
-
 
-
 
-
===Mix for PCR (x76 tubes)===
 
-
 
-
 
-
25µl Quick load 2x
 
-
1µl Oligo F
 
-
1µl Oligo R
 
-
23µl H2O
 
Line 123: Line 114:
* 50µl of Mix PCR by tubes
* 50µl of Mix PCR by tubes
* 1 clone
* 1 clone
-
* Programme : 105°C -  for 29 cycles : 95°C; 5' - 95°C; 30" - 55°C; 30" - 72°C; 1,5'
+
* Program : 105°C -  for 29 cycles : 95°C; 5' - 95°C; 30" - 55°C; 30" - 72°C; 1,5'
===Results===
===Results===
 +
* '''PCR 1 : J23109 (A)'''
-
{|Paris_PCR_0| Title = J23109 (A)
+
{|border="1" Paris_PCR_1| Title = J23109 (A)
-
|PCRs setting
+
|'''PCRs setting'''
|
|
|Quick Load
|Quick Load
|25µL
|25µL
 +
|
|
|
|Expected size
|Expected size
Line 142: Line 135:
|v_oligoF
|v_oligoF
|10µM 1µL
|10µM 1µL
 +
|1000pb
 +
|-
 +
|55°C
|
|
-
|35pb
+
|n_oligoR
 +
|19 VR
 +
|v_oligoR
 +
|10µM 1µL
 +
|Sucess
 +
|-
 +
|Time Elongation
 +
|
 +
|Water
 +
|23µL
 +
|
 +
|
 +
|Yes
 +
|-
 +
|1m30'
 +
|
 +
|DNA
 +
|Toothpick of J23109
 +
|
 +
|
 +
|Image see bands 1 to 8
 +
|-
 +
|Number cycles
 +
|
 +
|
 +
|
 +
|
 +
|
 +
|[[Image:080717-1.png|thumb|J23109]]
 +
|-
 +
|29
 +
|
 +
|
 +
|
 +
|
 +
|
 +
|Ladder band 0
 +
|}
 +
 
 +
 
 +
* '''PCR 2 : E0030 (K)'''
 +
 
 +
{|border="1" Paris_PCR_2| Title = E0030 (K)
 +
|'''PCRs setting'''
 +
|
 +
|Quick Load
 +
|25µL
 +
|
 +
|
 +
|Expected size
 +
|-
 +
|Annealing
 +
|
 +
|n_oligoF
 +
|18 VF2
 +
|v_oligoF
 +
|10µM 1µL
 +
|723pb
|-
|-
|55°C
|55°C
Line 151: Line 204:
|v_oligoR
|v_oligoR
|10µM 1µL
|10µM 1µL
 +
|Sucess
 +
|-
 +
|Time Elongation
|
|
 +
|Water
 +
|23µL
 +
|
 +
|
 +
|Yes
 +
|-
 +
|1m30'
 +
|
 +
|DNA
 +
|Toothpick of E0030
 +
|
 +
|
 +
|Image see bands 9 to 16
 +
|-
 +
|Number cycles
 +
|
 +
|
 +
|
 +
|
 +
|
 +
|[[Image:080717-1.png|thumb|E0030]]
 +
|-
 +
|29
 +
|
 +
|
 +
|
 +
|
 +
|
 +
|Ladder band 0
 +
|}
 +
 +
 +
* '''PCR 3 : pSB1A3 (A)'''
 +
 +
{|border="1" Paris_PCR_3| Title = pSB1A3 (A)
 +
|'''PCRs setting'''
 +
|
 +
|Quick Load
 +
|25µL
 +
|
 +
|
 +
|Expected size
 +
|-
 +
|Annealing
 +
|
 +
|n_oligoF
 +
|18 VF2
 +
|v_oligoF
 +
|10µM 1µL
 +
|
 +
|-
 +
|55°C
 +
|
 +
|n_oligoR
 +
|19 VR
 +
|v_oligoR
 +
|10µM 1µL
|Sucess
|Sucess
|-
|-
Line 158: Line 271:
|Water
|Water
|23µL
|23µL
 +
|
|
|
|Yes
|Yes
|-
|-
|1m30'
|1m30'
 +
|
|DNA
|DNA
-
|Cycles= 29  
+
|Toothpick of pSB1A3
-
|n_oligoR= 19 VR
+
|
-
|v_oligoR= 10µM 1µL
+
|
-
|water= 23µL
+
|Image see bands 1 to 8
-
|DNA= toothpick of J23109
+
|-
-
|Size expected= 35 pb
+
|Number cycles
-
|Success= YES
+
|
-
|Image=
+
|
-
|Band=1-8
+
|
 +
|
 +
|
 +
|[[Image:080717-2.png|thumb|pSB1A3]]
 +
|-
 +
|29
 +
|
 +
|
 +
|
 +
|
 +
|
 +
|Ladder band 0
 +
|}
 +
 
 +
 
 +
* '''PCR 4 : C0179 (A)'''
 +
 
 +
{|border="1" Paris_PCR_4| Title = C0179 (A)
 +
|'''PCRs setting'''
 +
|
 +
|Quick Load
 +
|25µL
 +
|
 +
|
 +
|Expected size
 +
|-
 +
|Annealing
 +
|
 +
|n_oligoF
 +
|18 VF2
 +
|v_oligoF
 +
|10µM 1µL
 +
|
 +
|-
 +
|55°C
 +
|
 +
|n_oligoR
 +
|19 VR  
 +
|v_oligoR
 +
|10µM 1µL
 +
|Sucess
 +
|-
 +
|Time Elongation
 +
|
 +
|Water
 +
|23µL
 +
|
 +
|
 +
|Yes
 +
|-
 +
|1m30'
 +
|
 +
|DNA
 +
|Toothpick of C0179
 +
|
 +
|
 +
|Image see bands 9 to 16
 +
|-
 +
|Number cycles
 +
|
 +
|
 +
|
 +
|
 +
|
 +
|[[Image:080717-2.png|thumb|C0179]]
 +
|-
 +
|29
 +
|
 +
|
 +
|
 +
|
 +
|
 +
|Ladder band 0
 +
|}
 +
 
 +
 
 +
* '''PCR 5 : C0079 (K)'''
 +
 
 +
{|border="1" Paris_PCR_5| Title = C0079 (K)
 +
|'''PCRs setting'''
 +
|
 +
|Quick Load
 +
|25µL
 +
|
 +
|
 +
|Expected size
 +
|-
 +
|Annealing
 +
|
 +
|n_oligoF
 +
|18 VF2
 +
|v_oligoF
 +
|10µM 1µL
 +
|876pb
 +
|-
 +
|55°C
 +
|
 +
|n_oligoR
 +
|19 VR
 +
|v_oligoR
 +
|10µM 1µL
 +
|Sucess
 +
|-
 +
|Time Elongation
 +
|
 +
|Water
 +
|23µL
 +
|
 +
|
 +
|Yes
 +
|-
 +
|1m30'
 +
|
 +
|DNA
 +
|Toothpick of C0079
 +
|
 +
|
 +
|Image see bands 1 to 8
 +
|-
 +
|Number cycles
 +
|
 +
|
 +
|
 +
|
 +
|
 +
|[[Image:080717-3.png|thumb|C0079]]
 +
|-
 +
|29
 +
|
 +
|
 +
|
 +
|
 +
|
 +
|Ladder band 0
 +
|}
 +
 
 +
 
 +
* '''PCR 6 : R0079 (A)'''
 +
 
 +
{|border="1" Paris_PCR_6| Title = R0079 (A)
 +
|'''PCRs setting'''
 +
|
 +
|Quick Load
 +
|25µL
 +
|
 +
|
 +
|Expected size
 +
|-
 +
|Annealing
 +
|
 +
|n_oligoF
 +
|18 VF2
 +
|v_oligoF
 +
|10µM 1µL
 +
|277pb
 +
|-
 +
|55°C
 +
|
 +
|n_oligoR
 +
|19 VR
 +
|v_oligoR
 +
|10µM 1µL
 +
|Sucess
 +
|-
 +
|Time Elongation
 +
|
 +
|Water
 +
|23µL
 +
|
 +
|
 +
|Yes
 +
|-
 +
|1m30'
 +
|
 +
|DNA
 +
|Toothpick of R0079
 +
|
 +
|
 +
|Image see bands 9 to 16
 +
|-
 +
|Number cycles
 +
|
 +
|
 +
|
 +
|
 +
|
 +
|[[Image:080717-3.png|thumb|R0079]]
 +
|-
 +
|29
 +
|
 +
|
 +
|
 +
|
 +
|
 +
|Ladder band 0
 +
|}
 +
 
 +
 
 +
 
 +
* '''PCR 7 : E1010 (K)'''
 +
 
 +
{|border="1" Paris_PCR_7| Title = E1010 (K)
 +
|'''PCRs setting'''
 +
|
 +
|Quick Load
 +
|25µL
 +
|
 +
|
 +
|Expected size
 +
|-
 +
|Annealing
 +
|
 +
|n_oligoF
 +
|18 VF2
 +
|v_oligoF
 +
|10µM 1µL
 +
|801pb
 +
|-
 +
|55°C
 +
|
 +
|n_oligoR
 +
|19 VR
 +
|v_oligoR
 +
|10µM 1µL
 +
|Sucess
 +
|-
 +
|Time Elongation
 +
|
 +
|Water
 +
|23µL
 +
|
 +
|
 +
|Yes
 +
|-
 +
|1m30'
 +
|
 +
|DNA
 +
|Toothpick of E1010
 +
|
 +
|
 +
|Image see bands 1 to 8
 +
|-
 +
|Number cycles
 +
|
 +
|
 +
|
 +
|
 +
|
 +
|[[Image:080717-4.png|thumb|E1010]]
 +
|-
 +
|29
 +
|
 +
|
 +
|
 +
|
 +
|
 +
|Ladder band 0
 +
|}
 +
 
 +
 
 +
* '''PCR 8 : B0034 (A)'''
 +
 
 +
{|border="1" Paris_PCR_8| Title = B0034 (A)
 +
|'''PCRs setting'''
 +
|
 +
|Quick Load
 +
|25µL
 +
|
 +
|
 +
|Expected size
 +
|-
 +
|Annealing
 +
|
 +
|n_oligoF
 +
|18 VF2
 +
|v_oligoF
 +
|10µM 1µL
 +
|
 +
|-
 +
|55°C
 +
|
 +
|n_oligoR
 +
|19 VR
 +
|v_oligoR
 +
|10µM 1µL
 +
|Sucess
 +
|-
 +
|Time Elongation
 +
|
 +
|Water
 +
|23µL
 +
|
 +
|
 +
|Yes
 +
|-
 +
|1m30'
 +
|
 +
|DNA
 +
|Toothpick of B0034
 +
|
 +
|
 +
|Image see bands 1 to 7
 +
|-
 +
|Number cycles
 +
|
 +
|
 +
|
 +
|
 +
|
 +
|[[Image:080717-5.png|thumb|B0034]]
 +
|-
 +
|29
 +
|
 +
|
 +
|
 +
|
 +
|
 +
|Ladder band 0
 +
|}
 +
 
 +
 
 +
* '''PCR 9 : J23101 (A)'''
 +
 
 +
{|border="1" Paris_PCR_9| Title = J23101 (A)
 +
|'''PCRs setting'''
 +
|
 +
|Quick Load
 +
|25µL
 +
|
 +
|
 +
|Expected size
 +
|-
 +
|Annealing
 +
|
 +
|n_oligoF
 +
|18 VF2
 +
|v_oligoF
 +
|10µM 1µL
 +
|1000pb
 +
|-
 +
|55°C
 +
|
 +
|n_oligoR
 +
|19 VR
 +
|v_oligoR
 +
|10µM 1µL
 +
|Sucess
 +
|-
 +
|Time Elongation
 +
|
 +
|Water
 +
|23µL
 +
|
 +
|
 +
|Yes
 +
|-
 +
|1m30'
 +
|
 +
|DNA
 +
|Toothpick of J23101
 +
|
 +
|
 +
|Image see bands 2 to 7
 +
|-
 +
|Number cycles
 +
|
 +
|
 +
|
 +
|
 +
|
 +
|[[Image:080717-6.png|thumb|J23101]]
 +
|-
 +
|29
 +
|
 +
|
 +
|
 +
|
 +
|
 +
|Ladder band 0
 +
|}
 +
 
 +
 
 +
* '''PCR 10 : E0040 (A)'''
 +
 
 +
{|border="1" Paris_PCR_10| Title = E0040 (A)
 +
|'''PCRs setting'''
 +
|
 +
|Quick Load
 +
|25µL
 +
|
 +
|
 +
|Expected size
 +
|-
 +
|Annealing
 +
|
 +
|n_oligoF
 +
|18 VF2
 +
|v_oligoF
 +
|10µM 1µL
 +
|840pb
 +
|-
 +
|55°C
 +
|
 +
|n_oligoR
 +
|19 VR
 +
|v_oligoR
 +
|10µM 1µL
 +
|Sucess
 +
|-
 +
|Time Elongation
 +
|
 +
|Water
 +
|23µL
 +
|
 +
|
 +
|Yes
 +
|-
 +
|1m30'
 +
|
 +
|DNA
 +
|Toothpick of E0040
 +
|
 +
|
 +
|Image see bands 4 to 5
 +
|-
 +
|Number cycles
 +
|
 +
|
 +
|
 +
|
 +
|
 +
|[[Image:080717-7.png|thumb|E0040]]
 +
|-
 +
|29
 +
|
 +
|
 +
|
 +
|
 +
|
 +
|Ladder band 0
 +
|}
 +
 
 +
 
 +
 
 +
* '''PCR 11 : R0040 (A)'''
 +
 
 +
{|border="1" Paris_PCR_11| Title = R0040 (A)
 +
|'''PCRs setting'''
 +
|
 +
|Quick Load
 +
|25µL
 +
|
 +
|
 +
|Expected size
 +
|-
 +
|Annealing
 +
|
 +
|n_oligoF
 +
|18 VF2
 +
|v_oligoF
 +
|10µM 1µL
 +
|174pb
 +
|-
 +
|55°C
 +
|
 +
|n_oligoR
 +
|19 VR
 +
|v_oligoR
 +
|10µM 1µL
 +
|Sucess
 +
|-
 +
|Time Elongation
 +
|
 +
|Water
 +
|23µL
 +
|
 +
|
 +
|Yes
 +
|-
 +
|1m30'
 +
|
 +
|DNA
 +
|Toothpick of R0040
 +
|
 +
|
 +
|Image see bands 7 to 8
 +
|-
 +
|Number cycles
 +
|
 +
|
 +
|
 +
|
 +
|
 +
|[[Image:080717-7.png|thumb|R0040]]
 +
|-
 +
|29
 +
|
 +
|
 +
|
 +
|
 +
|
 +
|Ladder band 0
|}
|}

Latest revision as of 14:35, 31 July 2008

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Contents

Results of the transformations (Biobricks 2007)

Legende :

  • few , medium, a lot --> description of the number of CFU (Colony forming unit) produce by the transformation
  • A: Ampicilin, K: Kanamycin


Ref. Book Biobricks 2007 Antibio Results
- positive control A 2.800 CFU
- negative control A 0 CFU
2E_SP 1000 B0034 A few
10F_SP 1001 R0079 A medium
4F_SP 1013 C0079 K few
6H_SP 1001 C0179 A medium
6B_SP 1001 S03154 A 0 CFU
10F_SP 1002 J23101 A a lot
11F_SP 1002 J23109 A a lot
4C_SP 1000 R0040 A 2 CFU
2G_SP 1000 C0040 A 0 CFU
1C_SP 1008 PSB1A3 A few
7G_SP 1013 E1010 K medium
7E_SP 1001 E0040 A 2 CFU
2F_SP 1016 E0030 K a lot


Cloning of the transformations

  • Test 8 clones by Biobricks
  • Incubate O/N at 37°C on proper plates


PCR Screening of the transformants

Use of the same 8 clones of Biobricks that have been tested for cloning


Protocol

  • 50µl of Mix PCR by tubes
  • 1 clone
  • Program : 105°C - for 29 cycles : 95°C; 5' - 95°C; 30" - 55°C; 30" - 72°C; 1,5'

Results

  • PCR 1 : J23109 (A)
PCRs setting Quick Load 25µL Expected size
Annealing n_oligoF 18 VF2 v_oligoF 10µM 1µL 1000pb
55°C n_oligoR 19 VR v_oligoR 10µM 1µL Sucess
Time Elongation Water 23µL Yes
1m30' DNA Toothpick of J23109 Image see bands 1 to 8
Number cycles
J23109
29 Ladder band 0


  • PCR 2 : E0030 (K)
PCRs setting Quick Load 25µL Expected size
Annealing n_oligoF 18 VF2 v_oligoF 10µM 1µL 723pb
55°C n_oligoR 19 VR v_oligoR 10µM 1µL Sucess
Time Elongation Water 23µL Yes
1m30' DNA Toothpick of E0030 Image see bands 9 to 16
Number cycles
E0030
29 Ladder band 0


  • PCR 3 : pSB1A3 (A)
PCRs setting Quick Load 25µL Expected size
Annealing n_oligoF 18 VF2 v_oligoF 10µM 1µL
55°C n_oligoR 19 VR v_oligoR 10µM 1µL Sucess
Time Elongation Water 23µL Yes
1m30' DNA Toothpick of pSB1A3 Image see bands 1 to 8
Number cycles
pSB1A3
29 Ladder band 0


  • PCR 4 : C0179 (A)
PCRs setting Quick Load 25µL Expected size
Annealing n_oligoF 18 VF2 v_oligoF 10µM 1µL
55°C n_oligoR 19 VR v_oligoR 10µM 1µL Sucess
Time Elongation Water 23µL Yes
1m30' DNA Toothpick of C0179 Image see bands 9 to 16
Number cycles
C0179
29 Ladder band 0


  • PCR 5 : C0079 (K)
PCRs setting Quick Load 25µL Expected size
Annealing n_oligoF 18 VF2 v_oligoF 10µM 1µL 876pb
55°C n_oligoR 19 VR v_oligoR 10µM 1µL Sucess
Time Elongation Water 23µL Yes
1m30' DNA Toothpick of C0079 Image see bands 1 to 8
Number cycles
C0079
29 Ladder band 0


  • PCR 6 : R0079 (A)
PCRs setting Quick Load 25µL Expected size
Annealing n_oligoF 18 VF2 v_oligoF 10µM 1µL 277pb
55°C n_oligoR 19 VR v_oligoR 10µM 1µL Sucess
Time Elongation Water 23µL Yes
1m30' DNA Toothpick of R0079 Image see bands 9 to 16
Number cycles
R0079
29 Ladder band 0


  • PCR 7 : E1010 (K)
PCRs setting Quick Load 25µL Expected size
Annealing n_oligoF 18 VF2 v_oligoF 10µM 1µL 801pb
55°C n_oligoR 19 VR v_oligoR 10µM 1µL Sucess
Time Elongation Water 23µL Yes
1m30' DNA Toothpick of E1010 Image see bands 1 to 8
Number cycles
E1010
29 Ladder band 0


  • PCR 8 : B0034 (A)
PCRs setting Quick Load 25µL Expected size
Annealing n_oligoF 18 VF2 v_oligoF 10µM 1µL
55°C n_oligoR 19 VR v_oligoR 10µM 1µL Sucess
Time Elongation Water 23µL Yes
1m30' DNA Toothpick of B0034 Image see bands 1 to 7
Number cycles
B0034
29 Ladder band 0


  • PCR 9 : J23101 (A)
PCRs setting Quick Load 25µL Expected size
Annealing n_oligoF 18 VF2 v_oligoF 10µM 1µL 1000pb
55°C n_oligoR 19 VR v_oligoR 10µM 1µL Sucess
Time Elongation Water 23µL Yes
1m30' DNA Toothpick of J23101 Image see bands 2 to 7
Number cycles
J23101
29 Ladder band 0


  • PCR 10 : E0040 (A)
PCRs setting Quick Load 25µL Expected size
Annealing n_oligoF 18 VF2 v_oligoF 10µM 1µL 840pb
55°C n_oligoR 19 VR v_oligoR 10µM 1µL Sucess
Time Elongation Water 23µL Yes
1m30' DNA Toothpick of E0040 Image see bands 4 to 5
Number cycles
E0040
29 Ladder band 0


  • PCR 11 : R0040 (A)
PCRs setting Quick Load 25µL Expected size
Annealing n_oligoF 18 VF2 v_oligoF 10µM 1µL 174pb
55°C n_oligoR 19 VR v_oligoR 10µM 1µL Sucess
Time Elongation Water 23µL Yes
1m30' DNA Toothpick of R0040 Image see bands 7 to 8
Number cycles
R0040
29 Ladder band 0
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