Team:Stockholm/21 July 2010

From 2010.igem.org



Contents

Mimmi

flourecent pEX

colony PCR

Mix (ml) X8
sH2O 22.5 180 *Take two samples of respectively clone and add them to 10µl LB
F primer 1 8
R primer 1 8 pEX.BBa_J18930
DNA 0.5 4 pEX.BBa_J18931
tot 25µl pEX.BBa_J18932


2010-07-21 pEX.BBa J18930-32 clone A+B.jpg
Primers
pEX_VF conditions gel
pEX_VR time °C well sample
2m 94 1 ladder
30s 94 ) 2 BBa_J18930A
30s 60 > 30 cycles 3 BBa_J18930B
2m 72 ) 4 BBa_J18931A
10m 72 5 BBa_J18931B
oo 10 6 BBa_J18932A
7 BBa_J18932B
8 control
9 ladder


  • Well 4 + 7 no product?
Bad mixing?


over expression continue...

  • Sonicate the samples on ice. 4X30s with 30s pause in between.
  • Heat in 95°C for 1m
  • Centrifuge 13000rpm for 1m
  • Load 1µl on the comassie mini-gel with a special comb

--> Sample is slimey, DNA in it??

--> Hard to see the differences in the bands

LMWP

glycerol stock + preparation of plasmids

  • Start cultures from the PCR colonies
Mix
LB 12ml
Amp 8µl
colony 4µl
  • Keep in 30°C ON





The Faculty of Science at Stockholm University Swedish Vitiligo association (Svenska Vitiligoförbundet) Geneious Fermentas/ Sigma-Aldrich/