Laragen Sequencing Genotyping

Ready-To-Run Sequencing Services

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.

Sample Requirements:

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.

The turnaround time is overnight. Average reads can be up to 900 bp depending on the DNA quality and DNA and primer sequences.

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).

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

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. Please label on the side of plate. Don’t label on the top (seal or caps) of the plates.

If you aren't using our convenient free pickup service, ship your samples at room temperature to:

Laragen, Inc.
10755 Venice Blvd.
Los Angeles, CA 90034

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

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