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From 2010.igem.org

9/10/10

Lux Casette Right Promoter BBa_I1051 (2008) plate C002 well 3 – C, 2007, Plate 1 18P, 2006 DNA – 1 18 – P PLux/C1 Hybrid promoter BBa_K091107 – well 8 – P plate #2 yeast promoter BBa_K105024

Yeast promoter BBa_J63005 well 4 – A Plate 2 Yesterday (9/9), I streaked 2 plates containing LB/Agar. I streaked the transformed electro comp cells using the turntable method. Today no growth was found. Possible reasons for error I might have not cooled glass hocking sticks long enough.

- Today –

We need 1 PLux, 2 Yeast Transcription Factor 3 LuxR 4 Yeast Promoter 5 Yeast promoter that responds to yeast trans factor, we must look at Igems registry

Parts list for Igem 1 PLux BBa_K091107 10,12,21,23,25 Plate 2 Well 8 – P

2 P Yeast BBa_J63005 10,12,21,23 Plate 2 Well 4 – A

3 LuxR BBa_T9002 10,12,21,23,25 Plate 2 Well 9 – A 4 Gal

4 BBa_K105007 10,12,23,25 Plate 3 Well 9 – I

5 Yeast Prom. BBa_K207001 10,12,23,25 Harvard

We are going to electroporate parts 1 – 3, 4 today using the same protocol from yesterday.

1) I cleaned the hood using alcohol and all pippettors boxes and tools.

2) Set up Ice bath and placed all tools in Hood

3) the first part I will be electroporating is part # BBa_K091107 Plate 2 well 8 – P

4) I added 10 mL 18ohm H2O to well 8 – P and pipetted up and down 4 times then removed 2 mL from well and placed into 100 mL comp cells, Gal 4 transcription factor BBa_K105007 Plate 3 well 9 – I

5) First electro was 1.8 kv at 5.2 ms.

1A) we electroporate part # BBa_K105007 from plate 3 1.8 kv 4.2 ms well 9 – I following the same protocol from page 15.

2) we electroporated part # BBa_T9002 from plate 2 well 9 – A we added 2 mL of 18 ohm H2O to resuspend the DNA spl. electroporate 1.8 kv 4.2 ms

3) Now that they have sat for 1 hr. we will streak 2 plates for each part so “6” in all

4) with remainder I will add half each tube to 10 mL LB – Lennox 3 with Amp made fresh and 3 w/out Amp and put on shaker. -Making Amp-

250 mL treats 250 mL 10 mL per 10 mL .845 g / 30 mL

I added .6909 g to 5 mL 18 ohm H2O the dissolved I added 10 mL to “3” 15 mL Sterile Centrifuge tubes then added 10 mL LB – Lennox to “6” tubes then added 400 mL electroporated cells to them part1, part 3, part 4