From 2007.igem.org

Contents 1 Week 3 1.1 Tuesday 17/07 1.2 Wednesday 18/07 1.3 Thursday 19/07 1.4 Friday 20/07

Week 3

Tuesday 17/07

1. Restriction Digests (see Protocol 7):
   • BBa_J23119 (strong constitutive promoter) pSB1A2. 1 x NheI, 1 x PvuI. 680bp, 1430bp
   • BBa_R0062 (HSL and luxR) pBB1A2. 1 x EcoRI, 1 x PvuI. 1460bp, 660bp
   • BBa_J04500 (IPTG inducer and RBS) pSB1AK3. 1 x PvuII, 2 x PvuI. 2250bp, 1030bp, 730bp
   • BBa_p1010 (death gene) pSB3K3. 1 x BamHI, 2 x XhoI. 190bp, 2390bp,840bp.
   • BBa_E0040 (GFP no promoter) pSB1A2. 1 x Hime II, 1 x PvuI. 1630bp, 1170bp.
   • BBa_I52001 (6D) p5B4K5. 2 x AvaI, 1 x PvuI. 920bp, 1470bp, 2120bp.

Wednesday 18/07

1. Last night we diluted primers to 100 pmol/ul by adding the amount of dH2O given on the order sheet, and made working solutions to 10 pmol/ul (2 x 200 ul each - 20 ul stock and 180 ul dH2O).
2. Maija ran a gel of the restriction digests to check the sizes of the biobricks. These were the maps we made yesterday.
3. Maia is extracting DNA from 3 samples of ****, which were plated yesterday and grown in liquid media, using MoBio PowersoilTM DNA purification kit (see Protocol 8); only change was to shorten "shaking" time from 10 to 2 minutes.
4. PCR trial run using Reddymix and Touch 2 done on:
   • pGLTUR: XylR_prefix / XylR_suffix, Pr_prefix / Pr_suffix, Pr_prefix / XylR_suffix, Pu_prefix_EM / Pu_suffix_EM.
   • pQF52 plasmid: DntR_prefix / DntR_suffix
   • DmpR WT/24: DntR_prefix / DntR_suffix
   • Biobricks: R0062, E0040, J04500, J23119, p1010, I52001.
   • See Protocol 9 for PCR.

Thursday 19/07

1. Maia redid PCR on pGLTUR and pQF52 using Reddymix and a gradient program (See Protocol 9) to minimise the non-specific amplification evident in previous gels. New DntR suffix primer has arrived. Used the following primer sets.
   • XylR prefix and XylR suffix
   • Pr prefix and Pr suffix
   • Pr prefix and XylR suffix
   • Pu prefix and Pu suffix
   • DntR prefix and DntR suffix 2

Friday 20/07