The first primer for primer walking is the vector. Then if we want to continue the primer walking, we have to select the next primer.
Right now, we are using several other software to finish selection octamer primer from the octamer primer library.
Sequence Methods:
Automsated sequencing reactions were performed using ABI BigDye Terminator Cycle Sequencing Ready Reaction kit with AnpliTaq DNA Polymerase. Sequencing reactions were precipitated, pellets were resuspended by buffer, and was loaded onto a sequencing gel. The data was collected by an ABI PRISM 377 DNA Sequencher.
Using Sequencher 3.0 (GeneCodes, Inc.), the location of ocatamers identified by this comparison was presented on the sequence, using alignment settings that requirea 100% match and a 8 base overlap. After the positions of the octmaer matches were identified, the information was exported to a Microsoft Excel spreadsheet for further analysis.
After that, we have to analysis of template secondary structure. The template DNA sequence was analyzed for potential hairpin structure using OLIGO 5.0. After the location of the octamer was identified, a 40 base region of the template (centering on the ocatamer) was analyzed for potential hairpin structures.
