Team:Paris/August 7

From 2008.igem.org

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|colspan="6"|[[Image: KR000118_gel1.jpg|thumb|'''Gel 1 : L100-L101''']]
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|colspan="3"|PCR8_’’’L126(1-6)’’’
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|colspan="6"|[[Image: KR000120_gel3.jpg|thumb|'''Gel 3 : L120-L122''']]
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|colspan="6"|[[Image: KR000122_gel4.jpg|thumb|'''Gel 4 : L123-L126''']]
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|colspan="3"|PCR5_’’’L126(7-8)’’’
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|align="center"|'''Expected size'''
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|align="center"|'''Measured size'''
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|colspan="6"|[[Image: KR000087_3.jpg|thumb|'''Gel 3 : L106-L107''']]
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|colspan="3"|[[Image: KR000123_gel5.jpg|thumb|'''Gel 5 : L126''']]
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|colspan="6"|[[Image: KR000089_4.jpg|thumb|'''Gel 4 : L108.1-L108.2''']]
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Revision as of 18:36, 7 August 2008

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Contents

Result of the isolation of colonies

E0240 and pSB3K3

E0240 and pSB3K3 are ok : there is a lot of single colonies
Two colonies are picked for an overnight culture at 37°C, 225 rpm, in order to extract plasmids (MiniPreps)

S120 and S121

S120 and S121 : there is a problem, there is nothing on the plates. We have to check whether those strains are really resistant to Amp.
We checked in the strain library, actually the strains do not carry any resistance cassette. We plated them once again on petri dishes with LB without antibiotics

Results of the PCR we did last night

PCR with gradient to amplify pflhDC
Standart PCR to amplify pflgA, pflgB and pflhB




Transformations

Protocol

Use of TOP10 chemically competentcells

  • Defroze competent cells on ice during 5'
  • Add 5µl of DNA Ligation in 50µL of competent bacterias (or 1µL for the positive control puc19)
  • Incubate 30' on ice
  • Heat-shock the cells during 30" at 42°C without shaking
  • Put 2' on ice
  • Add 250µL of pre-warmed SOC medium (4°C)
  • Incubate 1h at 37°C under shaking (225rpm)
  • Spin at 5.000rpm during 30"
  • Remove 150µL of supernatant
  • Resuspent the pellet in the 150µL left
  • Spread on adequated plates
  • Incubate O/N at 37°C


List of the Ligation Transformation

Name Description Antibio
Ligation
L128 J61002-pFlgA
D136 (FV) - D132 (FI) 		Amp
L129 J61002-pFlgB
D136 (FV) - D133 (FI) 		Amp
L130 J61002-pFlhB
D136 (FV) - D134 (FI) 		Amp
L131 J61002-pFlhDC
D136 (FV) - D135 (FI) 		Amp
Control
Control 1 D136 Amp
Positive control pUC19 Amp


PCR Screening of Ligation Transformants of 1st August

Use of 8 clones of Ligation transformants for screening PCR


Protocol of screening PCR

  • Mix
Name Vol (µl) Concentration
Quick Load 25µl 2X
OligoF_VF2 (O18) 1µl 10µM
OligoR_VR (O19) 1µl 10µM
water 23µl


  • 50µl of Mix PCR by tube/clone
  • one toothpick of each clone's colony by tube
  • Program : Annealing 55°C - Time élongation 1'30" - Number cycle : 29


Conditions of electrophoresis

  • 10µl of ladder 1 kb
  • 10µl of screening PCR
  • migration ~30min at 100W on 1% gel


Results

PCR1_’’’L101(1-8)’’’ PCR2_’’’L102(1-8)’’’ PCR3_’’’L113(1-8)’’’ PCR4_’’’L114(1-8)’’’
Expected size Measured size Band Expected size Measured size Band Expected size Measured size Band Expected size Measured size Band
2-9 10-17 1, 3-9 10-17
Gel 1 : L100-L101
Gel 2 : L113-L114
PCR5_’’’L120(1-8)’’’ PCR6_’’’L122(1-4)’’’ PCR7_’’’L123(1-8)’’’ PCR8_’’’L126(1-6)’’’
Expected size Measured size Band Expected size Measured size Band Expected size Measured size Band Expected size Measured size Band
1,2, 4-9 10-13 2-8, 1 3-8
Gel 3 : L120-L122
Gel 4 : L123-L126
PCR5_’’’L126(7-8)’’’
Expected size Measured size Band
2-3
Gel 5 : L126
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