Raphaël

DNA precipitation in the gel extraction samples K081008 (RBS+LuxI) and R0062 (pLux) (concentration of the samples in DNA)

Initial volume : 50 µL ; Final volume : 5 µL

Protocol

1. Add 0.1 volumes of 3M Sodium Acetate solution to 1 volume of DNA sample.
2. Add 1ul Glycogen to the DNA sample.
3. Add 2 volumes of 95% EtOH to the DNA Sample.
4. Store the solution for 30 minutes at -80°C.
5. Centrifuge the solution at maximum speed for least 15 minutes.
6. Decant and discard the supernatant.
7. Air-dry the pellet for 10-15 minutes at room temperature until all liquid is gone.
8. Resuspend in desired volume of water or buffer

Gel electrophoresis (agarose 1,0% w/v, EtB) (check the presence of DNA in the concentrated gel extraction samples)

Protocol (5 wells)

• 10 µL of ladder 1Kb (new ladder)
• nothing
• 5 µL of concentrated gel extraction sample (K081008 (RBS+LuxI)) + 1 µL of loading buffer 6x
• nothing
• 5 µL of concentrated gel extraction sample (R0062 (pLux)) + 1 µL of loading buffer 6x

<a href="/wiki/Image:DNAcheck_150710.jpg" class="image" title="Image:DNAcheck_150710.jpg"><img alt="Image:DNAcheck_150710.jpg" src="/images/4/4b/DNAcheck_150710.jpg" width="356" height="404" border="0" /></a>
There's no DNA visible in the gel ! (a negligible amount of DNA in our gel extraction samples or no DNA at all ?)

Aleksandra and Léa

Miniprep - Kit Promega (5 mL of liquid culture)

• J23119 (strong promoter)
• J23110 (medium promoter)
• K081008 (RBS + LuxI)
• E0240 (RBS + GFP + terminator)
• R0062 (pLux)
• B0015 (double terminator)
• J37033 (RBS + LuxR)
• J31005 (CmR resistance)
• P1003 (KanR resistance)
• pi
• attC
• pSUlib EX-B0015-attC-S-mRFP1-P
• pBad18-IntI1
• pSWK attP-1E
• pTSA CX1 Int lambda

Gel electrophoresis (agarose 1,0% w/v, EtB) (check the concentration of DNA in the minipreps)

Protocol (12 wells)

• 5 µL of ladder 1Kb (new ladder)
• 7,5 µL attC + 1,5 µL of loading buffer 6x
• nothing
• 7,5 µL pSUlib EX-B0015-attC-S-mRFP1-P + 1,5 µL of loading buffer 6x
• 7,5 µL J23119 (strong promoter) + 1,5 µL of loading buffer 6x
• 7,5 µL J23110 (medium promoter) + 1,5 µL of loading buffer 6x
• 7,5 µL J37033 (RBS + LuxR) + 1,5 µL of loading buffer 6x
• 7,5 µL R0062 (pLux) + 1,5 µL of loading buffer 6x
• 7,5 µL E0240 (RBS + GFP + terminator) + 1,5 µL of loading buffer 6x
• 7,5 µL K081008 (RBS + LuxI) + 1,5 µL of loading buffer 6x
• 7,5 µL B0015 (double terminator) + 1,5 µL of loading buffer 6x
• 7,5 µL pBad18-IntI1 + 1,5 µL of loading buffer 6x

Migration : 50V, 1h00
<a href="/wiki/Image:Miniprep_150710.jpg" class="image" title="Image:miniprep_150710.jpg"><img alt="Image:miniprep_150710.jpg" src="/images/0/05/Miniprep_150710.jpg" width="729" height="416" border="0" /></a>

Restriction digest 1h, 37°C

Using 4 µL of DNA :

• J23119 (strong promoter) cut by SpeI + PstI (Buffer 2)
• J23110 (medium promoter) cut by SpeI + PstI (Buffer 2)
• K081008 (RBS + LuxI) cut by EcoRI + SpeI (Buffer 2)
• E0240 (RBS + GFP + terminator) cut by XbaI + PstI (Buffer 3)
• R0062 (pLux) cut by SpeI + PstI (Buffer 2)
• B0015 (double terminator) cut by EcoRI-HF + XbaI (Buffer 4)
• J37033 (RBS + LuxR) cut by XbaI + PstI (Buffer 3)

Using 10 µL of DNA :

• attC cut by SpeI + PstI (Buffer 2)
• EX-B0015-attC-S-mRFP1-P cut by XbaI + PstI (Buffer 3)

Aleksandra

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

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

Protocol (12 wells)

• 5 µL of ladder 1Kb
• 17 µL of digested DNA (attC cut by SpeI + PstI) + 3 µL of loading buffer 6x
• 17 µL of digested DNA (EX-B0015-attC-S-mRFP1-P cut by XbaI + PstI) + 3 µL of loading buffer 6x
• 17 µL of digested DNA (J23119 (strong promoter) cut by SpeI + PstI) + 3 µL of loading buffer 6x
• 17 µL of digested DNA (J23110 (medium promoter) cut by SpeI + PstI) + 3 µL of loading buffer 6x
• 17 µL of digested DNA (J37033 (RBS + LuxR) cut by XbaI + PstI) + 3 µL of loading buffer 6x
• 17 µL of digested DNA (R0062 (pLux) cut by SpeI + PstI) + 3 µL of loading buffer 6x
• 17 µL of digested DNA (E0240 (RBS + GFP + terminator) cut by XbaI + PstI) + 3 µL of loading buffer 6x
• 17 µL of digested DNA (K081008 (RBS + LuxI) cut by EcoRI + SpeI) + 3 µL of loading buffer 6x
• 17 µL of digested DNA (B0015 (double terminator) cut by EcoRI-HF + XbaI) + 3 µL of loading buffer 6x
• 17 µL of digested DNA (attC cut by SpeI + PstI) + 3 µL of loading buffer 6x
• 17 µL of digested DNA (EX-B0015-attC-S-mRFP1-P cut by XbaI + PstI) + 3 µL of loading buffer 6x

Migration : 50V, 1h
<a href="/wiki/Image:Extractionbefore_biobricks_150710.jpg" class="image" title="Image:extractionbefore_biobricks_150710.jpg"><img alt="Image:extractionbefore_biobricks_150710.jpg" src="/images/6/64/Extractionbefore_biobricks_150710.jpg" width="722" height="411" border="0" /></a> <a href="/wiki/Image:Extractionafter_biobricks_150710.jpg" class="image" title="Image:extractionafter_biobricks_150710.jpg"><img alt="Image:extractionafter_biobricks_150710.jpg" src="/images/0/0d/Extractionafter_biobricks_150710.jpg" width="723" height="396" border="0" /></a>
The migration is not correct because we have had water in the gel instead of TBE Buffer

Gel extraction - Kit Qiagen

vectors :

• attC cut by SpeI + PstI (wells 1+11)
• J23119 (strong promoter) cut by SpeI + PstI
• J23110 (medium promoter) cut by SpeI + PstI
• R0062 (pLux) cut by SpeI + PstI
• B0015 (double terminator) cut by EcoRI-HF + XbaI

inserts :

• EX-B0015-attC-S-mRFP1-P cut by XbaI + PstI (wells 2+12)
• E0240 (RBS + GFP + terminator) cut by XbaI + PstI

Ligation (T4 ligase), 25 min at room temperature
5 µL insert + 0,5 µL vector

• attC cut by SpeI + PstI (vector) + EX-B0015-attC-S-mRFP1-P cut by XbaI + PstI (insert)
• R0062 (pLux) cut by SpeI + PstI (vector) + E0240 (RBS + GFP + terminator) cut by XbaI + PstI (insert)

Transformation (using 5 µL of ligation product)
Transformation into TOP10 - pSB AmpR with biobricks - Plating 100µL of each on the plates (Amp) - Incubation overnight :

• Product of ligation attC + EX-B0015-attC-S-mRFP1-P -> Transformation didn't work
• Product of ligation R0062 (pLux) + E0240 (RBS + GFP + terminator) -> Transformation didn't work
• Reference plasmid pUC19 -> Transformation worked

Raphaël

Restriction digest 1h, 37°C then overnight at -20°C

Using 4 µL of DNA :

• J23119 (strong promoter) cut by SpeI + PstI (Buffer 2)
• J23110 (medium promoter) cut by SpeI + PstI (Buffer 2)
• K081008 (RBS + LuxI) cut by EcoRI + SpeI (Buffer 2)
• E0240 (RBS + GFP + terminator) cut by XbaI + PstI (Buffer 3)
• R0062 (pLux) cut by SpeI + PstI (Buffer 2)
• B0015 (double terminator) cut by EcoRI-HF + XbaI (Buffer 4)
• J37033 (RBS + LuxR) cut by XbaI + PstI (Buffer 3)

Using 10 µL of DNA :

• attC cut by SpeI + PstI (Buffer 2)
• EX-B0015-attC-S-mRFP1-P cut by XbaI + PstI (Buffer 3)