BigDye® Terminator v3.1 Cycle Sequencing Kit provides everything we need in order to generate a sequence all we need to do is add one primer and the DNA template. These reagents are suitable for performing fluorescence-based cycle sequencing reactions on single-stranded or double-stranded DNA templates, on polymerase chain reaction (PCR) fragments, and on large templates.
First, you must prepare your template by performing a DNA extraction and purifying your template. The purification will be done in a QIAquick column and it will remove dNTPs and primers from your PCR reactions that created your amplicon. Determine the quality of your DNA by running a gel- electrophoresis and a Spectrophotometry of your sample.
The thermos cycler begins the reaction by heating the two DNA strands to 90 Celsius in order to separate them. Then the primer anneals to the sequence at a lower temperature of 54 Celsius. Then cycler increases the temperatures to 72 Celsius and the DNA polymerase starts elongating the primer. If allowed to go to completion, a new strand of DNA would be the result. …show more content…
This is just like regular DNA, except it has no 3' hydroxyl group so once it's added to the end of a DNA strand, there's no way to continue elongating it.
Most of the nucleotides are regular ones, and just a small fraction of them is
First, you must prepare your template by performing a DNA extraction and purifying your template. The purification will be done in a QIAquick column and it will remove dNTPs and primers from your PCR reactions that created your amplicon. Determine the quality of your DNA by running a gel- electrophoresis and a Spectrophotometry of your sample.
The thermos cycler begins the reaction by heating the two DNA strands to 90 Celsius in order to separate them. Then the primer anneals to the sequence at a lower temperature of 54 Celsius. Then cycler increases the temperatures to 72 Celsius and the DNA polymerase starts elongating the primer. If allowed to go to completion, a new strand of DNA would be the result. …show more content…
This is just like regular DNA, except it has no 3' hydroxyl group so once it's added to the end of a DNA strand, there's no way to continue elongating it.
Most of the nucleotides are regular ones, and just a small fraction of them is