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Mutation detection for the,,,K- rasand P16 genes

as above, but for 40 cycles. Again, the amplicon was purified and subjected to the same restriction enzyme digest/purification. At this point the samples were run on the Agilent 2100 bioanalyzer and submitted for DNA sequencing. For the P16 gene, the final reaction concentrations were 0.1 M of each primer4 (Invitrogen), with 0.2 mM dNTPs (Invitrogen), 1 unit of Platinum Taq High Fidelity, 1X PCR buffer (Invitrogen), 3 mM magnesium sulfate, 3 % DMSO (Sigma), 10 ng of DNA template, and HPLC-grade water (Fisher) in a total volume of 50 l. An initial 3-minute denaturation at 95 C was followed by 40 cycles at the following conditions, again on ABIs 9700 thermal cycler: 95 C for one minute, 58 C for 1 minute, and 72 C for 1 minute. A ten minute final extension at 72 C was also incorporated. Amplicons were freed of excess primers and nucleotides using ExoSap (USB), analyzed on the Agilent 2100 bioanalyzer, and submitted for DNA sequencing. Sequencing reactions were purified/de-salted using Sephadex G50 (Sigma).
Analysis of PCR-products
Amplified products were electrophoresed on Agilents 2100 bioanalyzer. The DNA 1000 LabChip kit was used in accordance with manufacturers instructions. Briefly, 9 l of the gel dye mixture was added to the chip well labeled G. This was pressurized for one minute throughout the chip with the syringe attachment provided. Then 9 l of the gel dye mixture was added to the other two chip wells labeled with G. 1 l of ladder was added to the ladder well, followed by 5 l of the gel dye mixture. This was pipetted up and down several times to mix. 5 l of the markers were added to each of the twelve sample wells. 1 l of each sample was added to their corresponding wells on the chip. The chip was vortexed for one minute on the IKA vortex adapter provided at the recommended setting. The chip was
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