sequencing protocol

[quratulain]

sequencing protocol

thanks alot... by saying "A normal sequencing reaction includes only a single oligonucleotide to prime polymerization", you mean , we use only one primer?

[pmiguel]

.i am specially confused about the PCR reaction that includes bigdye that how this reaction do the amplification as no polymerase and other reagents are added?

I'll take one of your questions. "Big Dye" does include a polymerase, buffer, dNTPs and dye-labelled ddNTPs.

Please note that reaction, although performed on a thermal cycler, is not a PCR reaction. The "chain reaction" of PCR occurs because the products of one cycle become templates in the next. PCR leads to an exponential increase in target DNA amounts. A normal sequencing reaction includes only a single oligonucleotide to prime polymerization. By cycling the reaction you can linearly increase the number of product molecules produced. But these product molecules are not templates for subsequent cycles.

--
Phillip

[quratulain]

can anyone please illustrate the whole protocol of sanger sequencing in simple way that involves purification of PCR reaction, bigdye PCR reaction, purification...or please recommend any site which can clear the concepts....why each step is being done and the purpose of different reagents....i am specially confused about the PCR reaction that includes bigdye that how this reaction do the amplification as no polymerase and other reagents are added?