I am finishing a microbial genome-sequence by sequencing PCR-products (with chromosomal DNA as template) using the Sanger method and fluorescence labelled nucleotides.
quite often the PCR works, but i get no or only very short sequences in the susequent Sencuencing reactions, probably due to secondary structures or high gc-content.
Adding DMSO and Betain doesn't always work.
Now i thought that adding SSB(single-strand-binding)-Proteins should prevent the formation of secondary structures and enhance the Sequencing-reaction. The problem is i can find no up-to-date reference for using ssb-protieins in Sanger Sequencing, only in Pyrosequencing.
Does anybody have experience in this? Are ssb-Proteins fluorescent?
Since a collegue told me that adding BSA to the Sequencing reaction does not only disturb the evaluation of the Gel, but can actually harm the Sequencer i am a little scared to simply try it out.
SSB-Protein as additive to Sequence difficult Templates using Sanger Method