IGEM:Paris Liliane Bettencourt/Notebook/Paris 2010/2010/07/15

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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


DNAcheck 150710.jpg
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
Miniprep 150710.jpg

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
Extractionbefore biobricks 150710.jpg Extractionafter biobricks 150710.jpg
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)