PROTOCOL 010: DNA SEQUENCING BY NCSU GSL

(10/2012)

 

REAGENTS

 

DNA

 

Primer

 

Hi-Di formamide (for microsatellite/fragment analysis only)

 

Water

 

PROCEDURE

 

       For dsDNA plasmid:

 

500-600 ng dsDNA plasmid template

1 L 10 M primer

X L sterile water

12 L total volume

 

       For PCR Products:

 

As above, except use 10 ng template for every 100 bases of PCR fragment length, up to 80 ng.

 

       For Cosmid, BACs and Phage l DNA:

 

Submit 2-3 g DNA template and 5 L of 100 nM primer in a separate tubes.

 

       For microsatellites/fragment analysis:

 

Dilute PCR reactions and size standard in Hi-Di formamide, for a total volume of 10 to 15 L per sample.

 

Reagents should be assembled in 1.5 ml Eppendorf tubes.

 

SUBMISSION

 

All samples are to be submitted in person to GSL in accordance with their instructions. These can be found here: http://gsl.cals.ncsu.edu/sequencing/single-tube-sequencing/.

 

NOTES:

a)    Sample and primer names together should be less than 20 characters (max 10 each).

b)    Use the primers database coordinate in lieu of the primer name.

c)    Do not use characters other than a-z, A-Z, 0-9, and "-.

d)    Do not use any space within the sample and primer name.

 

Example: pGADT7smo-12J5 (14 conforming characters, sample is plasmid pGADT7smo, primer is 12J5 corresponding to T7 promoter primer)