How do I prepare my DNA templates for low volume (single reaction) sequencing on the ABI3730?

 

If you do not find answers here, please do not hesitate to contact us directly at 520-621-9184 or dnaseq@arl.arizona.edu.

Submitting DNA Templates:

Submit all samples in 1.5ml tubes. Write name of template exactly as it appears on the web site work order. On the side of the tube, write your name and the tracking number from the web site. Please note in the comments section of your work order if you anticipate difficult template (e.g. GC rich). If the template is to be used for more than one sequencing reaction, submit only one tube with enough DNA for all of the reactions.

Required Volumes for Submission:

Template- 8µL/reaction
Primer- 5µL/reaction

Sample Preparation for PCR products:

The most important factors are concentration and purity . We strongly recommend that all PCR products be prepared with commercially available PCR cleanup kits to remove excess primers, nucleotides, and buffers. The most commonly used PCR purification kit among our users is Qiagen's QIAquick PCR system.

Important! Do not use buffers containing EDTA (such as T.E.) to resuspend your templates. This will cause your sequencing reaction to fail! We recommend submitting samples in sterile ultrapure (MilliQ) water.

 

What are the ideal concentrations and quantities for templates and primers?

Ideal Concentrations:

template
concentration
*total ng per reaction

Plasmids (up to 10 KB including insert)

200ng/µL

500 ng

Plasmids (over 10 KB including insert

200-400 ng/µL

500-1000 ng

BAC DNA

1-2 µg/µL

5 ng

Cosmids, Phage DNA

1µg/µL

2.5 ng

PCR... Optimum concentration depends on length . Use the formula below:

Multiply the length of PCR product (Base pairs) x 0.02 = concentration (in ng/µL )

 

* Templates at optimal concentration use a standard 2.5ul of template per reaction. If template concentration is lower than ideal concentrations, we can use up to 8ul of template. When you submit your sample online, please enter correct concentrations. Our computer will then calculate how much template will be optimal in your reaction.

Primers... 3-5pmol/µL (3-5µM) Note: for a 20-mer primer this is 20ng/ul.