IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/07/08

From OpenWetWare

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

Aleksandra and Léa

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

  • BBa_J23119 - strong promoter - Amp resistance - 18 A plate 1 - pSB1A2
  • BBa_J23110 - medium promoter - Amp resistance - 20C plate 1 – pSB1A2
  • BBa_K081008 - RBS + LuxI - Amp resistance - 10L plate 2 - pSB1A2
  • BBa_E0240 - RBS + GFP + terminator – Amp resistance - 12M plate 1 - pSB1A2
  • BBa_R0062 - pLux – Amp resistance - 60 - plate 1 - pSB1A2
  • BBa_B0015 - double terminator – Amp resistance - 23L plate 1 - pSB1AK3
  • BBa_J37033 - RBS + LuxR – Amp resistance - 40 plate 2 - pSB1A2
  • BBa_P1003 – Make primers to amplify KanR w/o promoter - 5P plate 1 - pSB1A1

Protocol
1- Melt bugs on ice ~10' for single shot.
If necessary, aliquot bugs into 50 µl per reaction.
Flick tube, if cells move then they are melted.
2- Add DNA to cells.
Do NOT pump up and down! Elute DNA into bugs, keep micropipette plunger constantly depressed, swirl using pipette tip, remove micropipette.
In general, use 2 µl of a 20 µl ligation rxn, 2 µl of a 6 µl TOPO rxn, or 1 µL of plasmid DNA.
3- 15-30' on ice.
15' sufficient for Amp. 30' for Kan
Can be left on ice for a few hours, some people leave it overnight but it is not recommended.
Pre-warm 37C appropriate antibiotic plates
4- Heatshock 30" @ 42°C
Have seen 45" and 90" heatshocks, all work.
5- 2' on ice.
6- Add 150 µl S.O.C., recover 30-60' at 37°C with shake.
If Amp resistant DNA, plate immediately after SOC addition.
7- Plate


Raphaël and Léa

Miniprep - Kit Qiagen - Final volume : 50µL

  • pSUlib EX-B0015-attC-S-mRFP1-P
  • pBad18-IntI1
  • pSWK attP-1E
  • Pi1
  • pTSA CX1 Int lambda

Glycerol 15% stored at -20°C

  • pSUlib EX-B0015-attC-S-mRFP1-P
  • pBad18-IntI1
  • pSWK attP-1E
  • Pi1
  • pTSA CX1 Int lambda




Personal tools