Team:Paris/August 27

From 2008.igem.org

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(→Cloning of EnvZ* in pSB1A2)
(→Cloning of EnvZ* in pSB1A2)
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No correct clone
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<br>The 8 other clones were also screened.
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'''PCR'''
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elongation time: 2 min 30
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'''Electrophoresis'''
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{|border="1" style="text-align:center"
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|'''sample'''
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|1 kb DNA ladder
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|positive control
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|negative control
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|colspan="8"|'''EnvZ*-pSB1A2 ligation'''
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|100 bp DNA ladder
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|colspan="8"|1659 bp
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<br>
=Cloning of OmpR*=
=Cloning of OmpR*=

Revision as of 17:18, 27 August 2008

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Contents

Construction of pFlhB - mRFP Tripart (LVA+)

Aim : Construction of "pFlhB-RBS-mRFP-dbl ter" (pFlhB-I732078) Part icon regulatory.pngPart icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png We can only do the construction with mRFP Tripart (LVA+) because the stable strain with the Biobricks I732011 (mRFP Tripart LVA-) don't to growth.


Digestion

Measurement of the concentration of D187 purified

Protocol (it's same that for Miniprep)

Digestion Name Concentration (µg/mL) Ratio 260/280
D187

Ligation

Protocol

Ligation Name Vector Name Volume Vector (µL) Insert Volume Insert (µL)
L1 D187 D186
Control L1 D187 - -

Cloning of EnvZ* in pSB1A2

Transformation results

control insert / vector mass ratio
transformation with pUC19 transformation without plasmid ligation without insert 1,8 / 1 2,4 / 1 3,1 / 1
number of colonies many 0 0 8 6 2
number of clones picked up for screening 4 2 2

PCR screening

  • screening programm
  • elongation time: 2 min
  • number of cycle: 24
  • total volume reaction: 25 µL
  • primers used: O18 and O19
  • positive control: S158 (pSB3K3)
  • negative control: no template

Electrophoresis

Screening EnvZ.JPG
well n° 1 2 3 4 5 6 7 8 9 10 11 12
sample 1 kb DNA ladder positive control negative control EnvZ*-pSB1A2 ligation 100 bp DNA ladder
clone 1 2 3 4 5 6 7 8
expected size 1659 bp
measured size below 0,3 kb


No correct clone
The 8 other clones were also screened.

PCR elongation time: 2 min 30
Electrophoresis

well n° 1 2 3 4 5 6 7 8 9 10 11 12
sample 1 kb DNA ladder positive control negative control EnvZ*-pSB1A2 ligation 100 bp DNA ladder
clone 9 10 11 12 13 14 15 16
expected size 1659 bp
measured size


Cloning of OmpR*

Digestion

Determination of the concentration of DNA

We used the biophotometer

  • 5 µL of template DNA or 5 µL of EB buffer for th blank
  • 55 µL of pure water
Template DNA Concentration of DNA
PCR 147 150 µg/mL
PCR 148 101 µg/mL
MP 101.2 353 µg/mL

Name of the digestions

Name of the digestion Template DNA What's in? Enzymes used
D 188 PCR 148 OmpR* XbaI-PstI
D 189 MP 101.2 pSB1A2 XbaI-PstI

Protocol of digestion

  • D 188 : 3 µL of PCR 148
  • D 189 : 3 µL of MP 101.2
  • 3µL Buffer (n°2) 10X
  • 0.3µL BSA 100X
  • 22.7 µL of pure Water
  • 1 µL of each enzyme
  • Incubate during about 2h30 at 37°C
  • 20 minutes at 65°C

Cleaning of the digestion products

Standard protocol.

Ligation

Determination of the concentration of DNA

We used the biophotometer

  • 10 µL of template DNA or 10 µL of EB buffer for th blank
  • 50 µL of pure water


Template DNA Concentration of DNA
D 188 9 µg/mL
D 189 30 µg/mL

Protocol of ligation L171

  • 2 µL Ligase Buffer 10X
  • 1.5 µL D 189 (vector)
  • 5 µL D 188 (insert)
  • 11.5 µL pure Water (qsp 20 µL)
  • 1 µL T4 DNA ligase at 400 000 U/mL concentration
  • O/N at 16°C

Checking mutagenesis FliA

For this, i digested mutated FliA and non-mutated FliA with EcoRI and PstI and put in migration the digestion products running on gels.
Results : No digestion for the mutated sequence --> successful mission !

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