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Plasmids

Sequencing mini-prepped plasmids of size 3 Kb to 10 Kb is relatively straightforward.  Poor results are usually due to problematic sample, not our Sanger protocol, which is verified with positive controls on every sequencing plate.  See Results Analysis for how to find relevant control results.  Please confirm template quality and quantity before submitting your samples.  The below recommendations should yield high-quality sequence reads.  If not, we can help you troubleshoot the problem.

 

PCR Products

Sequencing a PCR product is much trickier than sequencing a plasmid.  Poor results are common because samples are frequently contaminated with confounding variables like primer pairs and/or secondary PCR products.  Moreover, PCR product sequencing requires VERY LITTLE DNA, at concentrations well-below the lower limit of quantification with NanoDrop.  Often, even 40 cycles of PCR yields less DNA than can be quantified accurately with NanoDrop.

 


I. Quantity


Please do not trust your quantity measurements unless you have confirmed the quality of the template, as shown below.

 

II. Quality


I. Quantity


We HIGHLY recommend quantifying yield via fluorometric assay (such as Qubit or SYBR).

 


 II. Quality


 


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