Gentra Puregene Blood Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Frozen samples should be thawed and equilibrated to room temperature (15–25°C) before beginning the procedure.

Upstream tips
Frozen samples should be thawed and equilibrated to room temperature (15–25°C) before beginning the procedure.

Publication protocol

DNA PCR and Direct Sequencing for Detection of CEBPα Mutations. Genomic DNAs were extracted from frozen bone marrow mononuclear cells collected at both diagnosis and AML transformation by using a DNA extraction kit (Puregene Gentra System, Minneapolis, MN) according to manufacturer's instruction. The PCR reaction was done in a mixture containing 100 ng genomic DNA, 200 μmol/L deoxynucleotide triphosphate, 1× Gold PCR buffer, and 1 unit AmpliTaq Gold polymerase (Applied Biosystems, Foster City, CA), with two overlapping pairs of primers: PP1F 5′-TCGCCATGCCGGGAGAACTCTAAC-3′ and PP1R 5′-CTGGTAAGGGAAGAGGCCGGCCAG-3′, and PP2F 5′-CCGCTGGTGATCAAGCAGGA-3′ and PP2R 5′-CACGGTCTGGGCAAGCCTCGAGAT-3′, which cover the entire coding region of human CEBPα (14), on a DNA thermal cycler (Applied Biosystems 9600) using a program consisting of 35 cycles at 94°C for 60 seconds, 62°C for 40 seconds, and 72°C for 90 seconds, with an initial preheating at 95°C for 10 minutes and a final step for 10 minutes at 72°C. PCR products were electrophoresed on agarose gels, purified with a MinElute gel extraction kit (Qiagen, Hilden, Germany), and sequenced using BigDye Terminators and AmpliTaq DNA polymerase FS (Applied Biosystems 3730) in both directions. Samples with abnormal sequencing results were subjected to PCR and sequencing again with alternative four pairs of primers as previously described (15), except for a modification in the second region (323 bp) using primers of BF 5′-CGAGTTCCTGGCCGACCTGT-3′ (nucleotide position 225-244) and BR 5′-GCTGGTAAGGGAAGAGGCCG-3′ (nucleotide position 528-547; Genbank accession no. NM_004364.2).

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Manufacturer protocol

Download the product protocol from Qiagen for Gentra Puregene Blood Kit below.

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