Team:Paris Liliane Bettencourt/Notebook/2010/07/09/

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Aleksandra, Léa, Raphaël and Théotime

DNA Gel electrophoresis (agarose 1,0% w/v) (Dosage of DNA before the restriction digest)

2 µL of EtBr was added in the gel

Protocol (6 wells)

2 µL of minipreped DNA (Qiagen - attC) + 2 µL of loading buffer 2x
5 µL of minipreped DNA (Qiagen - attC) + 1 µL of loading buffer 6x
10 µL of minipreped DNA (Qiagen - attC) + 2 µL of loading buffer 6x
2 µL of minipreped DNA (Qiagen - dbter+attC+mRFP) + 2 µL of loading buffer 2x
10 µL of minipreped DNA (Qiagen - dbter+attC+mRFP) + 2 µL of loading buffer 6x
10 µL of ladder 1Kb

Migration : 50V, 30 min

10 µL of minipreped DNA corresponds to approximately 125ng of DNA (see fluorescence of the 3Kb band of the ladder)

Restriction digest

pSUlib - attC KanR cut by SpeI and PstI
pSUlib EX-B0015-attC-S-mRFP1-P KanR cut by XbaI and PstI

2 conditions for each plasmid :
condition 1 : Using 2 µL of DNA (add 40 µL of water)
condition 2 : Using 5 µL of DNA (add 37 µL of water)

Final volume : 50 µL

42-X µL distilled water
5 µL buffer 10x
0,5 µL BSA 100x
X µL minipreped DNA (roughly 500 nanograms)
1 µL restriction enzyme 1
1 µL restriction enzyme 2

Protocol
1- Add in order listed above.
2- Vortex the entire mixture for 2 sec and then spin down using a desktop microcentrifuge.
3- Put the reaction in the 37°C incubator for 2h.

DNA Gel electrophoresis (2 gels, agarose 1,0% w/v) (gel extraction for purification of DNA)

After migration, we made a bath with EtBr during 5-10 min.

Protocol (attC gel) (6 wells)

20 µL of digested DNA (attC - SpeI+PstI - condition 1) + 3 µL of loading buffer 6x
20 µL of digested DNA (attC - SpeI+PstI - condition 1) + 3 µL of loading buffer 6x
20 µL of digested DNA (attC - SpeI+PstI - condition 2) + 3 µL of loading buffer 6x
20 µL of digested DNA (attC - SpeI+PstI - condition 2) + 3 µL of loading buffer 6x
20 µL of digested DNA (attC - SpeI+PstI - condition 2) + 3 µL of loading buffer 6x
10 µL of ladder 1Kb

Migration : 50V, 30 min
<a href="/wiki/Image:Extractionbefore_attC_090710.jpg" class="image" title="Image:extractionbefore_attC_090710.jpg"><img alt="Image:extractionbefore_attC_090710.jpg" src="/images/0/04/Extractionbefore_attC_090710.jpg" width="374" height="409" border="0" /></a> <a href="/wiki/Image:Extractionafter_attC_090710.jpg" class="image" title="Image:extractionafter_attC_090710.jpg"><img alt="Image:extractionafter_attC_090710.jpg" src="/images/0/05/Extractionafter_attC_090710.jpg" width="361" height="413" border="0" /></a>
Extraction of the bands corresponding to the vector (P-plasmid-E-X-attC-S)

Protocol (RFP gel) (6 wells)

20 µL of digested DNA (RFP - XbaI+PstI - condition 1) + 3 µL of loading buffer 6x
20 µL of digested DNA (RFP - XbaI+PstI - condition 1) + 3 µL of loading buffer 6x
10 µL of ladder 1Kb
20 µL of digested DNA (RFP - XbaI+PstI - condition 2) + 3 µL of loading buffer 6x
20 µL of digested DNA (RFP - XbaI+PstI - condition 2) + 3 µL of loading buffer 6x
20 µL of digested DNA (RFP - XbaI+PstI - condition 2) + 3 µL of loading buffer 6x

Migration : 50V, 30 min
<a href="/wiki/Image:Extractionbefore_RFP_090710.jpg" class="image" title="Image:extractionbefore_RFP_090710.jpg"><img alt="Image:extractionbefore_RFP_090710.jpg" src="/images/f/fc/Extractionbefore_RFP_090710.jpg" width="363" height="418" border="0" /></a> <a href="/wiki/Image:Extractionafter_RFP_090710.jpg" class="image" title="Image:extractionafter_RFP_090710.jpg"><img alt="Image:extractionafter_RFP_090710.jpg" src="/images/2/28/Extractionafter_RFP_090710.jpg" width="380" height="416" border="0" /></a>
Extraction of the bands corresponding to the insert (X-dbter+attC+mRFP-S-P)

Gel extraction - Kit Qiagen

Ligation (T4 ligase)

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)
Ligation overnight in cold water

Protocol

Final volume : 20 µL
- 2 µl 10x Buffer
- 0.5 µl vector
- 1 or 5 µl insert
- 15,5 or 11,5 µl water (vector + insert + water = 17ul)
- 1 µl Quick ligase

Vortex the mixture
For each vector-insert pair, set up 4 ligations : 1) vector only control 2) vector + ligase control 3) vector + 1 µl insert + ligase 4) vector + 5 µl insert + ligase

Antoine

Results of transformation from yesterday

Colonies on all plates -> storage at 4°C before overnight culture

Théotime and Raphaël

Overnight culture 37°C

10mL LB + Amp

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