MinElute Gel Extraction Kit (250)

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Construction of shotgun libraries from BAC DNA: Large-scale preparations of BAC DNA were carried out using the Large-Construct kit from Qiagen (Qiagen GmbH, Hilden, Germany). After sonification and enzymatic repair of the ends, fragments of desired size (usually 1.2 - 1.5 kb) were isolated from a 1% preparative agarose gel using the MinElute Gel Extraction kit (Qiagen) and inserted into a Sma I-digested and alkaline phosphatase-treated pUC19 vector88. Ligation was carried out with the Rapid Ligation kit (Roche) according to the manufacturer’s protocol. The ligation mixture was then desalted using a QIAquick kit (Qiagen) according to the instructions of the supplier with the exception of the elution step performed with distilled H2O. 1/10 volume of the eluted DNA was used for transformation of competent Escherichia coli DH10B cells using a Genepulser II device (Bio-Rad). 1 ml Luria Bertani (LB) medium was added and incubated for 1 h at 37°C. 1/200 and 1/20 volumes of the transformed cells were plated onto Petri dishes containing LB agar, ampicillin, X-Gal and isopropylthiogalactoside (IPTG)88 and grown overnight at 37°C to determine the yield of recombinant clones. Usually the transformation frequency exceeded 108 transformants per µg vector DNA and the white:blue ratio was approximately 10:1 or better.


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