Will Notebook2

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Wideloache 15:45, 15 January 2009 (EST)

I ran my digestion of J61002-J23100 (E/P) on a 0.8% gel and got the following result:

0.8% gel. Lane 1: J61002-J23100 (E/P)

I expected to see a band at around 900, which would have been the RFP insert, but since the vector was fairly faint as well, it probably just wasn't bright enough to see. I excised the band at around 2000 bps which should be pSB1A2 vector cut with E/P.

I also ran my digestions of LacIX86-pLux' and LacII12X86-pLuxLas on a 2.0% gel and got the following result.

2.0% gel. Lane 1: LacIX86-pLux' (E/P). Lane 2: LacII12X86-pLuxLas (E/P)

This was what I expected to see, and I excised the lowest band on each gel (expected size was 93 bps).

I did a gel purification of all 3 excised gel fragments, and then I ligated pSB1A2 with each of the pLux promoter parts. I also did a negative control ligation of pSB1A2 + H20. I expect to have the unsuccessful ligation be red, making the verification of successful ligations easier. I plated on LB Amp plates after transforming into JM109 cells.

Wideloache 20:36, 13 January 2009 (EST)

Today was my first day back in the lab of the semester. The first thing that I needed to do was to get the two pLux promoters that had been synthesized by GeneArt into pSB1A2. I had done an (E/P) digestion of pLux' and pLuxLas last semester that seemed to be successful. However, when I tried to transform into pSB1A2, I had a low colony yield. I decided to make a fresh stock of pSB1A2 digested with E/P. I used the part J61002-J23100. Cutting this with E/P woudl result in a pSB1A2 vector with a RFP expression insert. In this way I could screen out unsuccessful ligations. In addition to running this digestion O/N, I also digested pLux' and pLuxLas with E/P from the minipreps of LacIX86-pLux' and LacII12X86-pLuxLas.

Make GenR plates (15 ug/mL)
Miniprep pLas' and pLasLux
Get GFP plasmid to put 4 promoters in front of.