Topo ta

Amplicons of MMTV env-pol DNA using the primers described below were ligated into TOPO-TA (Invitrogen), transfected into E. coli on agar plates, and grown o/n at 37 °C (TOP 10 cells; Invitrogen). Positive colonies were identified by blotting and probing with the MMTV specific sequences above. They were then grown o/n in LB broth. DNA was extracted using QI prep Spin Miniprep Kit (Qiagen Sciences, Germantown, MD) and then sent to the Cornell University Life Sciences Core Laboratories Center (Ithaca, NY) for DNA sequencing using overlapping primers: BR-7321F-5′-CCA ATA CAA AAC TGG TCC CTA-3′ and BR-7507R-AAA TCC CAA AGT AAC CCA AGG-3′, BR-7462F-5′-GGG TGA GTT TTT CTC CAA AAG G-3′ and BR-8070R-5′-AAT CAA AGC AGA TAT GCC CAG-3’ [32 (link)]. The respective primers used were termed the “sequencing” primers, and dTTP rather than dUTP was used to generate these amplicons (Fig. 1).

Genomic DNA was isolated from leaves, endosperm, and embryo at 6 or 7 days after pollination using a CTAB procedure. Bisulfite treatment was performed using the MethylCode Bisulfite Conversion Kit (Invitrogen, Life Technologies Corporation) or BisulFlash DNA Bisulfite Conversion Easy Kit (Epigentek Group Inc.) following the manufacturer’s protocols. 2 μl bisulfite treated DNA was used in PCR reactions with 2.5 U ExTaq DNA polymerase (Takara) and 0.4 μM primers using the following cycling conditions (95°C 3 minutes, 40 cycles of [95°C for 15 seconds, 50°C for 30 seconds, 72°C for 45 seconds], 72°C for 5 minutes). PCR products were gel purified, cloned using a TOPO-TA (Invitrogen) or CloneJet (Life Technologies) PCR cloning kit and individual colonies were sequenced. Sequences were aligned using SeqMan and methylation was quantified using CyMate [50 (link)].