"

From 2014.igem.org

25^th June

• Digestion PCR / Backbones
  
  Finale digestion concentration - 50ng/µl
  H2O 7,5µl
  Buffer 2µl
  Enzyme 0,25µl
  Enzyme 0,25µl
  Concentration : 10µl -> 100ng/µl
  20µL -> 50ng/µl
  



• Digestion ELP-20 -40 and -60
  
  20 (16/06/14) -> 685,7ng/µl
  Vi=100*10/685,7=1,46µl +8,54µl H2O
  40 (23/06/14) -> 282,5ng/µl
  Vi=100*10/282,5=3,53µl +6,47µl H2O
  60 (23/06/14) -> 366,3ng/µl
  Vi=100*10/366,3=2,73µl +7,27µl H2O
  
  pSB6A1 -> 100ng/µl
  (2013 plaque2 -> 2L) Vi=10µl
  
  (pSB1C3)x3 -> 100ng/µl
  (well 17O plate 4 ; 2014 well 23P plate 3 ; well 23O plate 3) Vi=10µl
  



• Cloning n°1
  o pSB6A1
  o insert 20 PCR of 10/06/14 -> 446,7ng/µl
  Vi=100*10/446,7=2,24µl +7,76µl H2O
  

• Purification ELP-20, -40,- 60 with Glycerol by SDS-PAGE-Laemmli 1X
  
  To heat sample 5min at 85°C.
  
  ELP-20 :
  No-induced -20, sample 2h20 and sample 4h - sample buffer 1X, beads and vortex. We put 6µl of sample
  
  Purif 1 and 2 - We put 15µl (5µl samples + 10µl sample buffer 1X)
  
  Migration 180V 500mA 1h10
  
  We use Prestained protein ladder EuroMedex #06P-0111
  ELP 20 = 8685,4 Da
  ELP 40 = 17035,4 Da
  ELP 60 = 25849,3 Da