IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/06/30

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6/30/10
Nanodropped PAL PCR cleanup—23.3 ng/ul

digestion

Changed digestion reaction since we only have ~400 ng DNA and protocol calls for 500 ng

Incubate at 37&#186; 15 min

Inactivate 80 &#186; 20 min

Ligation:

RT 10 min

80&#186;C 20 min

Transformation:

100 ul competent cells + 2 ul PAL/pSB1A2 ligation. Electroporate, add 1mL LB, 1 hour recovery at 37&#186;C. No growth after 10 hours.

Colony Boil: MG1655

Gradient PCR Each reaction gets 4 ul primers, 35.6 ul mastermix (which includes everything except primers). Ars and PAL primers at 10uM.

1.	ArsB 45&#186;C

2.	ArsB 48&#186;C

3.	ArsB 51&#186;C

4.	ArsB 54&#186;C

5.	ArsB 57&#186;C

6.	ArsB 60&#186;C

7.	ArsB 63&#186;C

8.	PAL

9.	PAL (-) no primers

Program:

gel

1% TAE, 175V NO BANDS! Only ladder showed up!

digestions 37&#186;C incubation 15 min

80&#186;C inactivation 20 min

ligation

RT 10 min

80&#186;C 20 min

transformation

100 ul cells, 2 ul ligation (J23100-gas vesicles, J23119-gas vesicles) electroporate, add 1mL LB, 1 hour recovery at 37&#186;C. got no growth after 10 hours.

Gel

80V

Ligation

incubated at room temp for 10 min, inactivated at 80°C for 20 min.

Warmed Amp/LB plate in 37°C room at 6:50 PM

7:15 PM

Transformation 100 ul competent cells and 2 ul ligation product in cuvette. electroporate and add 1 mL LB for recovery at 37°C 1 hour.

plated 20 ul on 1 half of Amp/LB plate, 200 ul on other half.


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