DNA Sequencing: Sample Preparation Recommendations

Please read the following important sequencing sample preparation guidelines to insure the highest quality and fast turn-around of your data. Note that the short FAQ below answers the most common questions regarding our sequencing services. For more information, please download and read our Sequencing FAQ.

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Concentration Requirements

Sample Type or Sequencing Primer	Concentration	Volume Required
Sequencing Primer	10 picomole/ul	2 ul/reaction, 10 ul minimum
PCR Product	10 ng/ul	25 ul
Small Insert Plasmid	50 ng/ul	25 ul
BAC/YAC/Cosmid	250 ng/ul	25 ul

Common Questions:

1. What should I re-suspend my prepared plasmid DNA in?
   
   Large and small insert plasmid DNA should be re-suspended in distilled water or 1x Tris.
   
   
2. How should I prepare my small or large-insert plasmid DNA?
   
   All plasmid DNA must be purified by alkaline lysis + ion-exchange or cesium gradient centrifugation for the best results.
   
   
3. How should my PCR Products look on a gel if I want to sequence them?
   
   PCR products should be a single band on a gel, though we have successfully sequenced products showing more than one band.
   
   
4. What do you mean by 'PCR Product Purification'?
   
   PCR Product Purification refers to the need to remove unincorporated dNTPs and primer(s) from the original PCR prior to sequencing which would otherwise interfere with the cycle-sequencing. The client may do this prior to shipping the samples to us, or have us do it by our recommended method, Exo/SAP.
   
   
5. What is the best method to determine DNA sample concentration?
   
   Concentration of DNA templates can be determined by the following methods, from most to least recommended:
   
   
   1. Agarose Gel
   2. Fluorimeter
   3. Spectrophotometer
      
      
6. What universal primers does Amplicon Express provide?
   
   Amplicon Express provides T7, T3, M13F, M13R, and many other 'universal' primers at no charge. We will also synthesize any vector primer for free when provided with the 5' to 3' sequence of the primer.
   
   
7. Where can I find more information?
   
   You can get the pdf version Sequencing FAQ(.pdf), view our Sequencing Troubleshooting page, or contact Jason Dobry for more details.