submission requirements

We recommend 2x amount of DNA in case we need to repeat the reactions

The quality of purified plasmid DNA is important to obtain good sequencing data. For best results DNA should be re-suspended in molecular biology grade water or Tris buffer. Avoid using EDTA which can inhibit the sequencing reaction. The purified plasmid DNA can be quantified by spectrophotometer. The optimal concentration of the purified plasmid template is 50-200 ng/µl.

The purity of the PCR product is crucial to obtaining good sequencing data. Any PCR primers and/or dNTPs remaining in the PCR product will adversely affect the quality of the sequencing. If the PCR product has a unique band, it can be purified by an enzymatic ExoSap reaction or commercial PCR purification kit. If the PCR product has more than one band, it should be run on the agarose gel to isolate the desired band for purification. The PCR products can then be quantified either by agarose electrophoresis or spectrophotometer. The optimal concentration of the PCR template is 20 ng/µl per KB (For example: optimal concentration of a 500bp PCR product would be 10 ng/µl).

It is necessary to measure the concentration of your DNA templates accurately by spectrophotometer. The primary factor contributing to poor sequencing results is inaccurate DNA concentration. So, please verify the concentration of your DNA template accurately.

DNA and Primer Separated

We recommend 2x amount of DNA in case we need to repeat the reactions

DNA and Primer Combined

We recommend 2x amount of DNA in case we need to repeat the reactions.

Example 1: If your 500bp PCR template and primer are at the concentration of 20ng/µl and 10pmol/µl, respectively, you need to add 3µl of PCR template and 2µl of primer to the 0.2 mL PCR 8-tube strip, and add 7µl of H2O to bring the total volume of 12µl.

Example 2: If your Plasmid template and primer are at the concentration of 100ng/µl and 10pmol/µl, respectively, you need to add 6µl of Plasmid template and 2µl of primer to the 0.2 mL PCR 8-tube strip, and add 4µl of H2O to bring the total volume of 12µl.

Use 0.2 mL PCR 8-tube strips or 1.5ml Epi tube. You can assign tube ID when you order online and it should match the label on your tubes. Ideally tube labels are short but unique enough to be distinguishable from other customer’s orders. The sample name you enter will be used to generate the name of the results file and can be whatever you wish. For orders with 16+ samples, strips are preferred.

Please submit your DNA and primers at the concentration according to the table above. The concentration of DNA and primers should be normalized and arrayed arranged vertically (A1-H1, A2-H2, etc.) on the 96 well plate as shown below. The plate should be sealed using strip-cap lids to prevent leakage and shipped overnight. Tube strips, while convenient, are hard to label and track, so for large orders (40+ samples) 96 well plates are preferred. Well fitting strip-cap lids will seal plates to prevent leakage and cross contamination. This is especially important for plates shipped overnight.

Depending on the specifics of your project, your libraries will require anywhere from 1ng to 1 ug of high quality DNA. For RNA libraries please submit RIN (or RQN) numbers along with your sample and its concentration. NGS is particularly sensitive to the quality of starting material. Please Contact your project manager today.

qPCR based assays: Please submit a minimum of 20 ng/uL sample per desired reaction (x2).

NGS based assays: Depending on the specifics of your project, your libraries will require anywhere from 1ng to 1 ug of high quality DNA. For RNA libraries please submit RIN (or RQN) numbers along with your sample and its concentration. NGS is particularly sensitive to the quality of starting material. Please contact Sequencing@retrogen.com to determine the best input for your project.

Sequence Verification: If submitting PCR products please see above for specific sample type to be submitted from sanger sequencing.