A. Amount of template needed per request
Please use the following template concentrations as a guideline for template submission.
PCR Product:
- 100-1000 bp: 5-20ng
(We use 20ng of DNA per reaction)
>1000 bp: 20-50ng (We use 50ng of DNA per reaction) - Single-stranded: 25-50ng (We cannot check the concentration of SS DNA. Please provide us with the concentration and the size of your template)
- Double-stranded: 150-300ng
(We use 150ng of DNA per reaction)
<10kb - Cosmid, BAC: 500ng-1ug (We use 1ug of DNA per reaction)
B. Concentration of DNA
Your template(s) should be submitted to us in clean HPLC grade water not TE or autoclaved water. While this does not ensure long term storage of the samples, it does maximize the potential of getting sequences. Typically, we would like the concentration of the template to be about 150 ng/uL for plasmids and about 15 ng/uL for PCR products. However, if you are not sure of the exact amount of template that you are submitting, then send it in 20 uL water and we’ll quantitate the sample for you.
NOTE: Use of TE or autoclaved water does not mean that you will have a problem, but they will definitely enhance any problems that already exist with your sample.
C. Age
Typically, your template should be no more than six months old.
D. Template Prep
There are several ways that a template can be prepared for sequencing. The first and probably easiest involves the use of a commercially available purification kit. Good results have been obtained from purification kits by Boehringer Manheim, Qiagen and Promega. It is important to remember that all of these kits can give quality results. As a result, you need to choose the kit that gives you the best results. Finally, CsCl banding or PEG ppt have had some success with sample preparation.
E. Problem Templates
For troublesome templates, a Microcon or comparable method of cleanup can be used to remove any contaminants that exist in the sample.
H. Quantitation of Template
Every double stranded template is quantitated in our facility using a fluorometer. If you are using a different method (ie. a UV/Vis spectrophotomer) to quantitate your samples, then you may want to send enough sample to calibrate your spec to ours.
We need to know if your template is GC or AT rich. This will allow us to adjust our quantitation value so that we do not underload or overload your template.
I. Shipping
How you ship your DNA samples is your choice. If they are in solution, ideally send them by priority mail (FedX, DHL, UPS, etc.) on some form of ice. Otherwise, send them dry using US mail or priority mail. However, we have noticed that some dried templates do not resuspend very well, so you may want to take this into consideration when choosing a shipping method.
Address:
The Sequencing and Synthesis Facility
University of Georgia
Riverbend Research Lab Room 161
110 Riverbend Rd
Athens, GA 30602