IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/07/14

From OpenWetWare

Jump to: navigation, search
Population counter Main project page
Previous entry      Next entry

Entry title

Léa

Restriction digest 1h, 37°C (10µL of minipreped DNA)
As none of the transformations worked, we want to make sure which step was faulty. We're checking if all of our restriction enzymes work properly.

  • E0240 - SpeI
  • E0240 - PstI
  • E0240 - EcoRI
  • E0240 - XbaI
  • E0240 - XbaI+PstI from 13/07
  • E0240 - SpeI+PstI

Gel electrophoresis (agarose 1,0% w/v, EtB) (verification of the restriction enzymes' activity and the presence of DNA in the gel extraction sample)

Protocol (8 wells)

  • 10 µL of ladder 1Kb
  • 7,5 µL of undigested plasmid (E0240) + 1,5 µL of loading buffer 6x
  • 7,5 µL of digested DNA (E0240 - SpeI) + 1,5 µL of loading buffer 6x
  • 7,5 µL of digested DNA (E0240 - PstI) + 1,5 µL of loading buffer 6x
  • 7,5 µL of digested DNA (E0240 - EcoRI) + 1,5 µL of loading buffer 6x
  • 7,5 µL of digested DNA (E0240 - XbaI) + 1,5 µL of loading buffer 6x
  • 7,5 µL of digested DNA (E0240 - XbaI+PstI) + 1,5 µL of loading buffer 6x
  • 7,5 µL of digested DNA (E0240 - SpeI+PstI) + 1,5 µL of loading buffer 6x
  • 7,5 µL of gel extraction DNA (E0240 - XbaI+PstI) + 1,5 µL of loading buffer 6x

Migration : 50V, 1h00
Image:digestionenzymecheck_130710.jpg
All the enzymes seem to work correctly. Our problem is probably the DNA yield after all the steps : not enough DNA extracted by minipreps -> no DNA visible by electrophoresis after gel extraction !


Raphaël

Transformation
Transformation into TOP10 - Plating 100µL of each on the plates (Amp) - Incubation overnight :

  • Product of ligation Plux(R0062) + RBS-GFP-term.(E0240) in Top10 cells from the NEW box
  • Product of ligation LuxI(K01008) + double term.(B0015) in Top10 cells from the NEW box
  • reference plasmid pUC19 in Top10 cells from the NEW box
  • reference plasmid pUC19 in Top10 cells from the OLD box


Transformation into TOP10 - Plating 100µL of each on the plates (Kan) - Incubation overnight :

  • Product of ligation attC + term.-attC-mRFP in Top10 cells from the NEW box


Léa
Liquid cultures
Starting 10ml liquid cultures of all biobricks and other parts that we need so as to do better minipreps this time and have more DNA -Incubation overnight at 37°C on a rack in LB media with correspondent molecule added:

  • J23119 (strong promoter) -amp
  • J23110 (medium promoter) -amp
  • k081008 (RBS + LuxI)-amp
  • E0240 (RBS + GFP + terminator) -amp
  • R0062 (pLux) -amp
  • B0015 (double terminator) -amp
  • J37033 (RBS + LuxR) -amp
  • J31005 (CmR resistance) -amp
  • P1003 (KanR resistance) -amp
  • pi -dT
  • attC -kan
  • pSUlib EX-B0015-attC-S-mRFP1-P -kan
  • pBad18-IntI1 -kan
  • pSWK attP-1E -kan
  • pTSA CX1 Int lambda -kan


Personal tools