From 2007.igem.org

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Protocols

References

Resources

Orders

Biobricks Used

Gels

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Contents 1 Week 3 1.1 Tuesday 17th July 2007 1.2 Wednesday 18th July 2007 1.3 Thursday 19th July 2007 1.4 Friday 20th July 2007

Week 3

Tuesday 17th July 2007

1. Restriction Digests of transformed BioBricks (see Protocol 7):

Label	BioBrick	Plasmid	Description	Enzymes	Expected Sizes
3/19A	BBa_J23119	pSB1A2	strong constitutive promoter	NheI, PvuI	680bp, 1430bp
1/9G	BBa_R0062	pBB1A2	HSL and luxR	EcoRI, PvuI	1460bp, 660bp
1/16P	BBa_J04500	pSB1AK3	IPTG inducer and RBS	PvuII, PvuI	2250bp, 1030bp, 730bp
4/11C	BBa_p1010	pSB3K3	death gene	BamHI, XhoI	190bp, 2390bp,840bp
1/5H	BBa_E0040	pSB1A2	GFP no promoter	Hime II, PvuI	1630bp, 1170bp
4/6D	BBa_I52001	p5B4K5	High copy death gene	AvaI, PvuI	920bp, 1470bp, 2120bp

Wednesday 18th July 2007

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 Pseudomonas Aeruginosa, 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 (see Protocol 9 for PCR).

Template	Primers
pGLTUR	XylR_prefix + XylR_suffix Pr_prefix + Pr_suffix Pr_prefix + XylR_suffix Pu_prefix_EM + Pu_suffix_EM
pQF52	DntR_prefix + DntR_suffix
DmpR WT/24	DntR_prefix + DntR_suffix

1. • Biobricks: [http://partsregistry.org/Part:BBa_R0062 R0062], [http://partsregistry.org/Part:BBa_E0040 E0040], [http://partsregistry.org/Part:BBa_J04500 J04500], [http://partsregistry.org/Part:BBa_J23119 J23119], [http://partsregistry.org/Part:BBa_p1010 p1010], [http://partsregistry.org/Part:BBa_I52001 I52001].

Thursday 19th July 2007

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 20th July 2007

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