Team:Stockholm/5 September 2010

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Latest revision as of 11:00, 26 October 2010

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Contents

Mimmi

MITF-M

Gel

well sample
1 1kb ladder
2 MITF-M 1
3 MITF-M 2
4 MITF-M 3
5 MITF-M 4
6 blank


  • nothing...
    • Make a new colony PCR with DreamTaq


SOD.his/his.SOD

  • Weigh the gel slices and add XP2


tube gel+tube gel XP2 tot
SOD.his 0.99g 1.45g 0.46g 460µl 920µl
his.SOD 0.99g 1.37g 0.38g 380µl 760µl


  • Incubate at 60°C for 7m or until the gel has melted


  • Follow the E.Z.N.A. gel extraction kit
    • Wash 2 times with SPW wash buffer
    • Incubate 1m and then eluate with 50µl dH2O


  • DNA conc.
    • not able to measure...


Ligation

pSB1C3 pEX Conditions
Mix (µl) x2 (µl) x2 time °C
vector 1 2 10m 22
DNA 14 13
5x buffer 4 4
T4 ligase 1 1
sH2O 0 0
tot 20µl 20µl



MITF-M

Colony PCR

Mix (µl) x6 Primers conditions
sH2O 17.25 103.5 pSB_VF2 time °C
dNTP 2.5 15 pSB_VR 2m 95
F primer 1 6 30s 95 )
R primer 1 6 30s 55 > 30 cycles
10x buffer 2.5 15 1m20s 72 )
DreamTaq 0.25 1.5 10m 72
DNA 0.5 6x0.5 oo 10
tot 25µl 150µl





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

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