( ution) allowed successful amplification...)Successful PCR amplification and subcloning of a GC-rich DNA fragment

Several primer pairs were designed to amplify a 4.5 kb Myb-INT fragment of the c-myb gene. The PCR template was plasmid DNA purified using the QIAGEN Plasmid Maxi Kit. Each PCR contained 300 ng template DNA, 2.5 mM MgCl2, 0.2 mM each dNTP, and 2.5 units DNA polymerase, in 1x PCR Buffer. PCR was carried out in the presence and absence of 1x Q-Solution. PCR products were purified using the MinElute PCR Purification Kit. For the cloning procedure, 225 or 550 ng purified PCR product was added to 50 ng pDrive Cloning Vector in 1x Ligation Master Mix, in a total volume of 10 l. Subsequently, a 3 l aliquot of the ligation reaction was used to transform one tube of QIAGEN EZ Competent Cells, which were then directly plated onto LB agar plates containing X-gal and IPTG for blue/white screening, and ampicillin at 30 g/ml. A second set of ligation reactions were carried out, and 3 l aliquots used to transform electrocompetent DH5-a E. coli. After transformation, bacteria were grown at 37C for 40 minutes before plating as above. White colonies derived from both ligation reactions were picked and grown overnight at 37C in LB medium containing ampicillin at 20 g/ml. Pure plasmid DNA containing the PCR insert was isolated using the

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