Genotyper 2

Cells were maintained in culture for no more than six passages. All cell lines were tested by PCR/IF for Mycoplasma presence. Head and neck CAL27 (mutp53H193L), A253 (p53-null), and lung H1299 (p53-null) cancer cell lines were cultured in RPMI medium (Invitrogen, Carlsbad, CA, USA); all media were supplemented with 10% (v/v) FBS (Invitrogen, Carlsbad, CA, USA). All cell lines were purchased from ATCC and were authenticated by STR (Short Tandem Repeats) genotyping with the Promega PowerPlex® 1.2 system and the Applied Biosystems Genotyper 2.0 software for analysis of the amplicons.

For genetic analysis, 28 SSRs that had been optimized previously to work in pooled samples of 15 individuals were selected (S2 Table). These SSRs gave good coverage of the maize genome with all linkage groups represented. Fragments (alleles) of each SSR were generated via PCR according to CIMMYT standard protocols [34 ]. Electrophoresis was conducted using an automatic capillary sequencer ABI 3100 (Applied Biosystems, Foster City, CA) to separate and size the fragments. Data were analyzed using the programs GeneScan ® 3.1 (PerkinElmer / Applied Biosystems, Foster City, CA) and Genotyper ® 2.1 (PerkinElmer / Applied Biosystems, Foster City, CA) to generate a data set of all fragments including size in base pairs, peak height (corresponding to intensity of the amplified fragment), and quality score.

Mature and aborted seeds were ground in Eppendorf tubes and soaked with pre-treatment solution for 24 h. Total DNA from seed tissues was then isolated using the Plant DNA Extraction Kit (Tiangen Biotech, Beijing, China) according to the manufacturer’s protocol.
Six SSR primers were selected and labelled with a fluorescent dye for the paternity analysis (Table 4) [47 -49 (link)]. Polymerase chain reaction (PCR) amplification was performed using an ABI 9700 thermal cycler in a 12.5-μL reaction mixture containing 30 ng of genomic DNA, 1× Taq buffer, 2.5 mM MgCl₂, 0.25 mM dNTPs, 0.20 μM of each primer pair and 0.5 units of Taq DNA polymerase. The amplification reaction was performed with the following two procedures: first, an initial denaturation step at 94°C for 5 min, followed by 10 cycles at 94°C for 30 s, T_m for 30 s (decreasing at n°C per cycle, n = (T_mmax – T_mmin)/10), 72°C for 90 s, 20 cycles at the annealing temperature and a final extension at 72°C for 10 min and second, an initial denaturation step at 94°C for 5 min, 35 cycles at 94°C for 30 s, T_m for 30 s, 72°C for 90 s and a final extension at 72°C for 10 min.
PCR products were electrophoresed on an ABI 3100 Genetic Analyser (Applied Biosystems, Foster City, CA, USA), and allele sizes were determined using the fragment analysis software packages GeneScan 3.0 and Genotyper 2.1 (Applied Biosystems).