Submission Requirements

**We recommend 2x the 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 (which can be performed by Retrogen for an additional charge) or a commercial PCR purification kit. If the PCR product has more than one band, it should be run on an 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.

If you place an order for sequencing with samples we already have, e-mail sequencing@retrogen.com with both the new order number and the order number which used the sample last.

Standard Service: DNA and Primer Separated

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

Pre-mixed Service: 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 0.2mL PCR 8‐tube strip, and add 7μL of H₂O to bring the total volume to 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 0.2mL PCR 8‐tube strip, and add 4μL of H₂O to bring the total volume to 12μL.

You can assign a 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 customers’ orders.

WRONG

1, 2, 3…

RS2023Aseq12345, RS2023Bseq12445, RS2023Cseq12545…

RIGHT

RS1, RS2, RS3…

DNA-A, DNA-B, DNA-C…

Each sequencing request is also required to have a sample name which will be used to create the file name of the result. The sample name has specific restrictions: the name should not be longer than 15 characters and can only contain legal characters. Legal characters are: a-z, A-Z, 0-9, “_” and “-”. Illegal characters are any other characters.

For small orders, you can use 1.5mL or 0.5mL microcentrifuge tubes. Screw-caps are time-consuming, so pop caps should be used. Excessive parafilm also takes time to remove and should be avoided.

For any orders with more than 16 samples, please use 0.2mL PCR 8‐tube strips. For larger orders (48 or more samples), please use a 96-well plate.

Please submit your DNA and primers at the concentration according to the tables above. The concentration of DNA and primers should be normalized and arranged vertically (A1‐H1, A2‐H2, etc.) on the 96-well plate as shown below. Always include the sample’s well coordinate in the tube label when submitting the order to ensure the correct sample is used for every reaction. Well-fitting strip cap lids will seal plates to prevent leakage and cross-contamination. This is especially important for plates shipped overnight.

Frozen Pellet:

In Tubes: With a single colony inoculated 1-5mL LB medium in a tube or flask. Total volume of the tube or flask should be at least 4 times the volume of the medium. Incubate for 12-16 hours at 37°C with vigorous shaking.

In 96-well block: With a single colony inoculated 1.3mL LB medium. Incubate the cultures for 20–24 h at 37°C with vigorous shaking. If using a plastic lid or adhesive tape to protect against spill-over, make sure the lid is porous to allow aeration – if not, use a needle to poke 2-3 holes above each well.

DO NOT ALLOW IT TO OVERGROW – cells begin to lyse and plasmid yields will be reduced.

Pellet cells by centrifugation for 5 min at 2100 x g, then invert to dispose of the media. Tap on a paper towel to remove any remaining media if needed.

Colonies (well isolated, not more than a few days old) or Glycerol stock:

Let us know what antibiotic resistance the plasmid contains.

For local pickup, pellets and glycerol stocks can be sent on ice. For overnight shipping, use dry ice.

Send one tube per PCR product to be sequenced (even if sequencing with multiple primers). We want at least 10µL of PCR product.

qPCR-based Assays: Please submit a minimum of 20 ng/µL sample per reaction (x2).

NGS-based Assays: Depending on the specifics of your project, your libraries will require anywhere from 1ng to 1µg 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.