Sanger sequencing

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How do I sequence large templates directly?

Here and there we do receive a request to sequence large templates (i.e. chromosomal DNA, BACs, cosmids) directly.

We can offer this service to you but similarly as for standard samples where short pcr products or plasmids serve as templates also here the key prerequisite is the template amount. For example, if you consider sequencing of bacterial chromosomal DNA having a size of approx. 3-4 Mb you must supply approx 3 μg of this DNA mixed together with 25 pmol of your sequencing primer in a final volume 10 μl. We sequence this sample by using a special protocol developed in house for large templates. If your chromosomal DNA is pure and everything proceeds as expected the result you will get will be unrecognizable from results obtained when sequencing pcr products.

 

Example output of a direct chromosomal DNA sequencing, bottom pane: raw data, upper pane: electropherogram with excellent sequence up to 1000 bp:

 

 

 

For cosmids or BACs similar rules apply. In summary, sequencing a large template means to provide a lot of it and use alternate sequencing strategy in our lab. The success is however in the hands of a person responsible for template prep. Also keep in mind that the risk of mispriming is high and a properly designed primer is undoubtedly a key prerequisite of this strategy.

Note: We do not offer the option to order this service on line, please contact us in case of interest. 

 

Richard Nádvorník, richard.nadvornik@seqme.eu

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