Laragen Sanger Sequencing Services

With over a decade of experience and thousands of successful projects, Laragen proudly offers DNA sequencing services that you can rely on. We use proven technology and provide secure access to your results through our web interface. Next day turnaround is standard, with same day service available upon request.

The key to having long sequencing reads is the quality and quantity of the DNA. Too much or too little DNA and dirty DNA is the main cause of the reaction failure We recommend the Qiagen miniprep kits to prepare plasmid DNA. Certain commercial kits did not work well with automated Sanger sequencing.

EDTA will inhibit DNA sequencing reaction. Don’t elute DNA into TE buffer. Use only water or Qiagen EB buffer (10 mM TrisHCl, pH 8.0). Average reads can be up to 1000 bp depending on the DNA quality and primer sequences.

For 96-well plates, please arrange samples by the columns, not by the rows:  You can use upload and import file functions in our DNALIMS system to upload the 96-well plate format. No spaces or illegal characters are allowed in the DNALIMS. Allowable characters include: a - z, A - Z, 0 - 9, and hyphens. You can use this Template for file upload. 

Please seal the 96-well plate with caps, foil tape or heat seal tape to avoid possible cross contamination and sample evaporation.

samplechart

If you are not using our convenient free pickup service, ship your samples at room temperature to:
Laragen, Inc.
10601 Virginia Ave.
Culver City, CA 90232


Ready-To-Run Sequencing Service

This is the most economical sequencing service. With ready to run sequencing services, you will set up your own sequencing reactions. We will clean up your reactions and load the samples to our ABI 3730XL.

Customers will perform their own sequencing reactions in 8 strip PCR tubes in 10 ul of reaction volume. We provide FREE Bigdye cleaning for ready-to-run samples. Please don’t label the PCR strips on the caps, use the side of the tubes.

To fill the ready-to-run samples in the DNALIMS, please choose the primer field as NA to avoid confusion.

We recommend using the following protocols to do sequencing reactions.

Reagents Amount
DNA plasmid (4to 6 kb) 200 ng
PCR (100 to 500 bp) 10 to 50 ng
Primer (10 uM) 1 ul
BigDye 3.1 1 ul
5x Sequencing Dilution Buffer 2 ul
Sequencing Grade Water q.s.
Total 10 ul

And the following thermo cycle condition (with AB9700 thermo cycler)

Cycles
Temperature
Time
1
96 C
1 minute
30
96 C
10 seconds
50 C
5 seconds
60 C
4 minutes
1
4 C
forever

Premix Sequencing Service

Premix sequencing service is designed for customers who want to save sequencing cost and do not want to set up their own sequencing reactions.

With this services, customers will measure the DNA concentration and mix 200 ng of plasmid DNA (4 to 6 kb, or more DNA if the plasmids are large) and 10 to 50 ng of PCR DNA (100 to 50 bp, 10 ng for every 100 bp of PCR fragment) with 10 pmole of sequencing primer in a total of 15 ul in 8 strip PCR tubes. Please use sequencing grade water. Please don't label the PCR strips on the caps. Please label on the side of the tubes.

Unpurified PCR Template Sequencing Service

Many commercial PCR purification products are not robust enough for DNA sequencing purpose. One of biggest challenges is that the recovery yield is quite variable with these commercial kits. Laragen has developed a proprietary PCR purification technique to overcome this low and uneven yield issue. This service is similar to the purified template sequencing service except we will purify PCR DNA for you.

With this service, customers will perform PCR reactions in 25 ul volume. Run 5 ul on an agarose gel. If there is a single band in the PCR product, send us the rest of the 20 ul PCR DNA. It will help us to have a gel image picture along with the samples. No purification is needed. Laragen will perform PCR purification. We also require 5 ul of 10 uM primer for each sequencing reaction. Please don’t label the PCR strips on the caps. Please label on the side of the tubes.

Purified Template Sequencing Service

By far, this is the most common service we provide. With this service, you provide us purified plasmid or PCR DNA templates with or without DNA concentration. Unlike most of our competitors, which depend on customer's DNA concentration, we measure DNA concentration of every single sample with our fluorometer. Then we will adjust the amount of DNA used per reaction based on the DNA concentration we measure. This procedure enables us to have good sequencing quality and long reads.

Customers can send us 10 to 15 ul of purified plasmid and PCR DNA either in 8 strip PCR tubes or in individual 1.7 ml Eppendorf tubes and 5 to 10 ul of 10 uM primer in separate tubes. Laragen provides free common sequencing primers. Please do not label the PCR strips on the caps. Please label on the side of the tubes.

RCA Sequencing Service

We will use the RCA technique to amplify the circular plasmids DNA from bacterial cells and sequence the amplified DNA directly. This technique is especially good for low copy number plasmids and will save you time to prepare the miniprep.

  • Low density cell culture: This is the most popular format. We accept 500 ul of 2x LB overnight culture at 37oC with shaking at 300 rpm in deep well culture blocks. The culture block should be sealed with airpore tape. For the shipping purpose, we recommend you send the cell pellets by centrifuging and decanting the culture medium. The pellets need to be sent on dry ice to keep frozen.
  • Glycerol stock: 50 to 100 ul of glycerol stocks can be sent frozen on dry ice in 96-well plates. Seal plate with foil tape or caps.
  • Bacterial colonies on agar plate: The colonies on the agar plate need to be well separated to enable us to pick a single colony. If you need the colonies back, please number the colonies so you can match the sequencing results with the colonies. Wrap the plates with parafilm.

Primer Walking Sequencing Service

Our primer walking service includes primer design and synthesis, sequencing reactions and sequencing assembly. Our low price primer walking sequencing services only charges for the cost of the primers and the purified template sequencing reactions.

Customers can send us 20 to 30 ul of purified plasmid and PCR DNA 1.7 ml Eppendorf tubes and 5 to 10 ul of 10 uM original primers (if any) in separate tubes. Please label on the side of the tubes. Laragen will design and order the necessary primers to finish the complete sequences. In most of cases, we can get 700 to 800 bp per primer read. To request this service, please type primer walking in the comment field when submitting the request form online.

Difficult Template Sequencing Service

Using Laragen's proprietary protocols and techniques, we can handle your difficult templates! It will expedite the sequencing results if you inform us if the sequence contains the following structures: shRNA, RNAi, hairpin, GC rich motif, or AT rich motif. Please check GC field in the online request form when submitting these sequences. Additional charges may apply to these templates.

Customers can send us 10 to 15 ul of purified plasmid and PCR DNA either in 8 strip PCR tubes or in individual 1.7 ml Eppendorf tubes along with 5 to 10 ul of 10 uM primer in separate tubes. Please don’t label the PCR strips on the caps. Please label on the side of the tubes.

Get a Quote

Laragen, Inc
10601 Virginia Ave, Culver City, CA90232
310-280-0804 info@laragen.com