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how to clone my insert DNA into vector? - (Aug/06/2008 )

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If the gene have many polymorphism is good idea to clone because you will have either form and when sequence you will have just one signal. If you are going to align the sequence with other sequence the aligment is easier. When sequencing directly the PCR when have polymorphism you will see 2 signals, so will be a little difficult to align specially if using the free tools in the internet because sometimes can't no discriminate if in the sequence instead of ATCG there is another letter as Y, N, etc.

About gel purification if have a single band there is no need to use this protocol is better to use a column spin. easier, no UV to the DNA, better yield, etc.


We routinely sequence gel-purified bands of PCR products without any problems -- in fact, they're usually our cleanest sequencing reactions. For cloning a gel-purified fragment, we add guanosine (0.28 g/L) to the gel and the TAE running buffer as a UV protectant, but haven't found that necessary for fragments recovered for sequencing, likely because the bases damaged from UV (if any) on any particular DNA molecule will be vastly outnumbered by the number of DNA molecules in the population that have not been damaged in that position, and thus such damage will not influence sequencing results. Besides, it's always a good idea to sequence in both directions, thus a T (that might be damaged by UV) on one strand is an A on the other...

Using the same reasoning, you can have a bit more confidence in the sequence acquired from sequencing a PCR product directly than you can from sequencing a single cloned PCR product, because all polymerases have a rate at which they will introduce an incorrect base. This rate, however, is low enough so that again, in the *population* of PCR amplicons being sequenced, the base at any given position is correct in the *vast* majority of cases, thus any errors won't show up in a sequencing reaction. However, once you clone a particular PCR amplicon out of the population, any errors of incorporation that happened to be on that particular amplicon are fixed, and your sequence will be incorrect at that position if you sequence that plasmid clone.

Unless there are multiple forms of the gene at the DNA level (post-transcriptional or post-translational modifications won't interfere, because you're sequencing a DNA amplicon), you shouldn't have a problem.


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