Mj mini personal thermocycler

Genomic DNA was isolated from individual clones. Cells were harvested by trypsinization, washed with PBS, centrifuged at 5000 rpm for 5’ at room temperature in a microcentrifuge, and the supernatant discarded to yield a pellet of approximately 0.5 million cells. The cells were resuspended in 200 ul of digestion buffer (50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 50 mM NaCl, 0.5% SDS, 0.5 μg/ul proteinase K (LifeTechnologies)) and incubated for 2 hr at 37C. An equal volume of digestion buffer without proteinase K was added, and the lysates were extracted twice with an equal volume of 1:1 phenol:chloroform (Sigma), and once with chloroform. DNA was precipitated by the addition of two volumes of 100% ethanol, collected by centrifugation and the pellet washed with 70% ethanol, before air-drying and resuspension in water. The DNA concentration was determined and 100 ng of genomic DNA was used as a PCR template with the following oligos (IDT):
5’ GCG AGT GAG GAG CAG ACC 3’
5’ GCT CCC AAA CCT CAT TTC AA 3’.
OneTaq 2X Master Mix (NEB) was used according to the manufacturer’s instructions and with the following program: 95C 2’ 1 cycle; 95C 30s, 54C 30s, 72C 1’ for 35 cycles; 72C 5’; 4C 15’, on an MJ mini personal thermocycler (BioRad). PCR products were isolated from 1.5% LMP/ 1X TBE agarose gel using the QIAquick gel extraction kit (Qiagen) and sequenced using the same PCR primers.

Total RNA was isolated from lung cancer cells using the RNAgents Total RNA Isolation System (Promega). First-strand cDNA was synthesized from 10 μg of total RNA using Superscript II reverse transcriptase (Invitrogen). The PCR primers include those for CCND1, 5′-CCCTCGGTGTCCTACTTCAAA-3′/5′ CCAGGTTCCACTTGAGCTTGT-3′ and c-Myc, 5′-CCTCAACGTTAGCTTCACCAA-3′/5′-TTTGATGAA GGTCTCGTCGTC-3′. PCR reactions were performed on an MJ Mini Personal thermo cycler (Bio-Rad) with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the internal control. The cycle conditions are available upon request.