From 2014.igem.org

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23^rd June

• PCR of ELP-40 and ELP-60
  
  => Dilution primer 100µM => 10µM
  Primer forward: 1µl primer + 9µl H2O (Tm=58°C)
  Primer reverse: 1µl primer + 9µl H2O (Tm=68°C)
  
  Test of different hybridation temperature for PCR: 59°C 61°C 63°C 65°C 68°C
  
  Mix for 11 tubes
  

  H2O	357,5 µl
  Buffer 5X	110 µl
  Primer	27,5 µl
  dNTP	11µl

  
  Thermal cycler :
  
  3min 95°C
  25 cycles : 30sec 95°C
  

  30sec at all temperatures tested
  3sec 72°C
  

  5min 72°C



• Digestion cloning 1 (psB6A1) miniprep of 10/06/14 and VPGXG-20 (PCR of 10/06/14)
  

  	Digestion plasmid	Digestion insert
  H2O	7,5µl	7,5µl
  10X buffer 4	2µl	2µl
  XbaI	0,25µl	0,25µL
  EcoRI	0,25µl	0,25µl
  DNA	10µl (*)	10µl (**)

  
  (*) With DNA = 6μl vector + 4μl H2O
  (**) With DNA= 0.56μl Miniprep product + 9,44μl H2O



• Digestion vector psB6A1 :
  
  
  

  	Digestion plasmid
  H2O	7,5µl
  10X buffer 4	2µl
  XbaI	0,25µl
  EcoRI	0,25µl
  DNA	10µl



• Digestion psB1C3 and PCR 40 and 60
  OD PCR tube 40= 282,5 ng/μl
  OD PCR tube 60= 366,3 ng/μl
  Tube 40 = 0,88 DNA +9,12µl H2O
  Tube 60 = 2,5 DNA + 7,5 µl H2O



Production
We want to do a preculture test to have a better production.