How are the cDNA libraries sequenced?
Each EST is generated by sequencing the 5′, and sometimes the 3′, end of an individual cDNA clone. CK Library: We sequenced all cDNAs that showed some pattern by in situ of embryos and some that were uniformly expressed, but none that were not expressed. Selected cDNAs were sequenced from both ends, and others were sequenced entirely using walk-in primers. Sequencing was done using either Pharmacia Autoread Sequencing kits with Cy-5-labelled primers and run on a Pharmacia ALF Express automated DNA sequencer, or using ABI Prism Dye Terminator Cycle Sequencing Ready Reaction kits with AmplitaqR DNA Polymerase and run on an ABI Prism 373 automated DNA sequencer. Universal or T7 primers were used to read the 5′ end and reverse or T3 primers were used to read the 3′ end of the cDNAs. All other libraries: We are currently using dye terminator sequencing (ABI Big Dye). The reactions are sequenced on ABI 377 automated DNA sequencers using the ABI 3.3 Sequence Analysis software.
Each EST is generated by sequencing the 5′, and sometimes the 3′, end of an individual cDNA clone. CK Library: We sequenced all cDNAs that showed some pattern by in situ of embryos and some that were uniformly expressed, but none that were not expressed. Selected cDNAs were sequenced from both ends, and others were sequenced entirely using walk-in primers. Sequencing was done using either Pharmacia Autoread Sequencing kits with Cy-5-labelled primers and run on a Pharmacia ALF Express automated DNA sequencer, or using ABI Prism Dye Terminator Cycle Sequencing Ready Reaction kits with AmplitaqR DNA Polymerase and run on an ABI Prism 373 automated DNA sequencer. Universal or T7 primers were used to read the 5′ end and reverse or T3 primers were used to read the 3′ end of the cDNAs. All other libraries: We are currently using dye terminator sequencing (ABI Big Dye). The reactions are sequenced on ABI 3730xl capillary DNA sequencers.
