Sequencing DNA

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At the MIT biopolymers facility and probably with most sequencing centers, when doing a run-off sequencing reaction, an extra (sometimes pretty high amplitude) 'A' peak is seen at the end before the template ends. The workers at the biopolymers facility seemed surprised when they were told about this, but they were able to find out that the sequenase enzyme used in the sequencing reaction is a genetically modified form of Taq. Therefore, it is most probable that the extra A is the template-independent A that Taq tends to add to the 3'-end of DNA.
At the MIT biopolymers facility and probably with most sequencing centers, when doing a run-off sequencing reaction, an extra (sometimes pretty high amplitude) 'A' peak is seen at the end before the template ends. The workers at the biopolymers facility seemed surprised when they were told about this, but they were able to find out that the sequenase enzyme used in the sequencing reaction is a genetically modified form of Taq. Therefore, it is most probable that the extra A is the template-independent A that Taq tends to add to the 3'-end of DNA.
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'''Q: The MIT sequencing center recommends an amount of template DNA for a sequencing reaction.  If I have a mixed sample of DNA (for example, two plasmids purified from a culture) and want some of it sequenced (for example, one of the plasmids), should I submit the recommended amount of total DNA or the recommended amount of the DNA of interest?'''<br>
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A: It's best to submit a pure sample...but if you cannot, I would make sure that you submit the required amount of the template that the primer that you are using will anneal to.
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Any other DNA will not get labeled with the fluorescent dyes that we use to call a sequence....and will hopefully be inert...it should be inert unless your primer also anneals to the contaminant DNA.
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The dynamic range of the DNAsequencer is between 10 to 1000 nanograms...so if you are in that ballpark it should work.

Revision as of 15:43, 1 March 2006

Specific Protocols

MIT:Sequencing BioBrick DNA -- protocol for DNA sequencing of BioBrick parts

Notes

At the MIT biopolymers facility and probably with most sequencing centers, when doing a run-off sequencing reaction, an extra (sometimes pretty high amplitude) 'A' peak is seen at the end before the template ends. The workers at the biopolymers facility seemed surprised when they were told about this, but they were able to find out that the sequenase enzyme used in the sequencing reaction is a genetically modified form of Taq. Therefore, it is most probable that the extra A is the template-independent A that Taq tends to add to the 3'-end of DNA.

Q: The MIT sequencing center recommends an amount of template DNA for a sequencing reaction. If I have a mixed sample of DNA (for example, two plasmids purified from a culture) and want some of it sequenced (for example, one of the plasmids), should I submit the recommended amount of total DNA or the recommended amount of the DNA of interest?

A: It's best to submit a pure sample...but if you cannot, I would make sure that you submit the required amount of the template that the primer that you are using will anneal to.

Any other DNA will not get labeled with the fluorescent dyes that we use to call a sequence....and will hopefully be inert...it should be inert unless your primer also anneals to the contaminant DNA.

The dynamic range of the DNAsequencer is between 10 to 1000 nanograms...so if you are in that ballpark it should work.

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