Reaction Dilution when you are performing your own cycle sequencing reactions;

	Reg. 1/4	1/8 rxn	1/16 rxn
Total Volume	5	10	10
BigDye	2	1	0.5
5X buffer	0	1.5	1.75
DNA	2	2	2
Primer	1	1	1
H2O	0	4.5	4.75

Notes:

If DNA or primer is not concentrated enough, add more DNA and less water.

If you are submitting samples for a cycle sequencing reaction, we will need the following amounts of DNA and primer:

Primer	3.2 pmoles
plasmid DNA	300 ng
DNA from PCR	3 ng for products under 200 bp 10 ng for products under 500 bp 20 ng for products under 1,000 bp 40 ng for products under 2,000 bp

DNA sequencing works well for PCR amplified products and is usually more effective than sequencing from a plasmid. The PCR product needs to be purified to remove primers and dNTPs. Some of the different methods for purification include; PrepEase, Exo/SAP, Qiagen, and agarose gel purification. Once clean, one effective way to determine DNA concentration of your purified template is to run it on an agarose gel and compare your band intensity with that of a DNA molecular weight standard with known concentrations.

A submission example; if you are submitting a 500bp PCR product for cycle sequencing, please submit at least 4 microliters containing 3.3 ng/μl cleaned PCR product, and in this same volume of liquid, 1.1 pmole/μl primer. We will then take 3 μl of this mix to add to our cycle sequencing mix.

Processing samples on the 454 Life Sciences DNA Sequencer
If you are planning on submitting samples for Next generation sequencing on the 454 Life Sciences machine, please contact Ed Wilcox (phone 422-3647; email DNASC@byu.edu). The following manuals detail some of the basic protocols in operation at our facility;

Operator's Manual
Titanium emPCR Method Manual
Titanium General Library Preparation Method Manual
Data Analysis Software Manual

Submitting samples for Parallab 350 Processing

Plates containing 95 samples can be submitted to the Parallab 350 workstation. We need a minimum of 4 such submissions for cost-effective use of BigDye on this machine (we distribute 2 μl of BigDye across an entire plate and can't afford to loose much to evaporation). The following is a format that works; One plate with a set of 95 primers at 12.5 pmoles primer/μl along with four plates of 95 templates each (we ask that you leave the H12 well empty for our control). Please note that any one well across these four plates of templates will be using the same primer for sequencing. There are other sample/primer combinations that will work when submitting to this workstation. The machine will mix 190 nl of BigDye (this is part of the charge you will be purchasing when using this machine) with 100 nl of primer and 210 nl of template (or 310 nl of primer and template premixed in a well). The machine performs the cycle sequencing and then cleans the reaction products for loading on the ABI 3730xl DNA Analyzer. This machine has 96 capillaries and they do not work independent of each other. Please call the DNASC at 422-3647 if you would like more information.