Team:Groningen/Notebook/Wetwork 20July2012


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Plan for today:

1. plasmid isolation from E.coli Dh5A

2. ligate alsT promoter and GFP into pSB1C3 and pSac-Cm (B. subtilis backbone)

3. E.coli transformation for both

1. Plasmid isolation pSB1C3+insert from iGEM Groningen 2010

E.coli DH5a strong magenta = insert device OK!

plasmid concentration: 129,1 ng/ul

Purification of PCR product using gel DNA extraction kit

diluted in 50ul TE (elution buffer)

concentration: 35,6 ng.ul

Ligation pAlsT-GFP into backbone pSB1C3 (and pSac-Cm done next week 23/07)

Cut parts with restiction enzymes

alsT: EcoRI + SpeI

GFP: XbaI + PstI

backbone: EcoRI + PstI

Digestion reaction and subsequently ligation of:

1) alsT150-GFP-BB

2) alsT250-GFP-BB

3) alsT300-GFP-BB

4) alsT500-GFP-BB

3. Transformation of E.coli DH5a

4x promoter alsT testing devices

1x negative control

= 5x LB plate + 15 ug/ml Cm

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