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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

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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