Team:Paris/August 27

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===Protocol of digestion===
===Protocol of digestion===
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 +
* 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===
===Cleaning of the digestion products===
==Ligation==
==Ligation==
===Determination of the concentration of DNA===
===Determination of the concentration of DNA===
===Protocol of ligation===
===Protocol of ligation===

Revision as of 13:15, 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 5 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

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

Ligation

Determination of the concentration of DNA

Protocol of ligation
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