Raphaël and Théotime

Miniprep - Kit Promega

• 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

Aleksandra

Restriction digest (EcoRI + PstI) 37°C, 2h

• Ligation product (see 09/07/2010):(P-plasmid-E-X-attC-S) + (X-dbter-attC-mRFP-S-P)1 µL
• Ligation product (see 09/07/2010):(P-plasmid-E-X-attC-S) + (X-dbter-attC-mRFP-S-P)5 µL

DNA Gel electrophoresis (agarose 1,0% w/v) (check the function of T4 ligase and the ligation product)
2 µL of EtBr was added in the gel

Protocol (7 wells)

• 5 µL of ladder 1Kb + T4 ligase (incubation 10 min at room temperature) + 1 µL of loading buffer 6x
• 5 µL of ladder
• 5 µL of ligation product (1 µL) digested by EcoRI and PstI + 1 µL of loading buffer 6x
• 5 µL of ligation product (5 µL) digested by EcoRI and PstI + 1 µL of loading buffer 6x
• 5 µL of ligation product (5 µL) non digested + 1 µL of loading buffer 6x
• 5 µL of ligation control (vector without insert) + 1 µL of loading buffer 6x
• 5 µL of minipreped DNA (attC) + 1 µL of loading buffer 6x

Migration : 50V, 1h30
<a href="/wiki/Image:Electrophoresis_120710.jpg" class="image" title="Image:electrophoresis_120710.jpg"><img alt="Image:electrophoresis_120710.jpg" src="/images/d/dc/Electrophoresis_120710.jpg" width="312" height="419" border="0" /></a>

Ligation (T4 ligase)

Ligation overnight in cold water
vector : P-plasmid-E-X-attC-S (pSUlib - attC KanR cut by SpeI and PstI)
insert : X-dbter+attC+mRFP-S-P (pSUlib EX-B0015-attC-S-mRFP1-P KanR cut by XbaI and PstI)

Stéphane and Raphaël

Restriction digest 45min at 37°C, then overnight at -20°C

• BBa_J23119 cut by EcoRI + SpeI -> wrong restriction enzymes ! (use SpeI + PstI)
• BBa_J23110 cut by EcoRI + SpeI -> wrong restriction enzymes ! (use SpeI + PstI)
• BBa_J37033 cut by XbaI + PstI
• BBa_R0062 cut by EcoRI + SpeI -> wrong restriction enzymes ! (use SpeI + PstI)
• BBa_E0240 cut by XbaI + PstI