Team:HokkaidoU Japan/Notebook/August11

From 2010.igem.org

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Revision as of 06:25, 20 September 2010

Notebook

LB Culture

  • For every 2 mL of LB added 2 uL of antibiotics
  • Transfered a colony to LB
  • One colony didn't grow well so we isolated another one
  • Prepared more tubes for mini preps int he future

glycerol Stock

  • Added 1 mL of 80% Glycerol to screw cap tube
  • Added 1 mL of cell and broth solution to Glycerol after 2h of incubation
  • Sored at -80℃

Ligation

DNA Preparation for Ligation

  • Used 49 uL of yesterdays PCR product
  • Removed primers via Microcon YM-10
    • Added 450 uL of TE to make final volume of 500 uL
    • Centrifuged at 10000G for more than an hour till all 4 samples volume was less then 45 uL
  • Measured the final amount of samples and added TE till all were 45 uL
    • Used 500 uL tubes

Ligation System

From PCR products we only used No.3 （1-23L（terminator）178 bp made on previous day

Tube No. １ ２ ３ ４
PCR products 1 uL 1 uL 1 uL 1 uL
10x H Buffer - 1 uL 1 uL 1 uL
DW 9 uL 7.5 uL 7.0 uL 7.5 uL
Xba1 - 0.5 uL 0.5 uL -
Pst1 - - 0.5 uL 0.5 uL
Restriction Enzyme Digestion 4℃ at 37C for 60 min
Restriction enzyme Inactivation at 60C for 15 min
ligation solution 10 uL 10 uL 10 uL 10 uL
Ligation at 16C for 30 min
6x SB 4 uL 4 uL 4 uL 4 uL

→1% Agarose Gel Electrophoresis in 1/2 TBE + EtOh

Electrophoresis

  • Performed electrophoresis for every digested sample, 1 through 4
  • Used marker pUC119/Hinf
  • DNA solution was too　diluted and no bands were visible
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