University of Washington Department of Biochemistry

DNA Sequencing Facility




Service II - Analysis Only Service

Preparing your samples for this service consists of 4 steps:

  1. Setting up reactions
  2. Cycle sequencing
  3. Removing unincorporated dye
  4. Drying down and freezing prior to submitting samples

1. Setting up Reactions (Why 1/2 reactions are not better than 1/4 or 1/8 reactions for 3730XL)

Reagent
1/4 Reaction
(Recommended)
1/8 Reaction
(Possible when template is of very high quality)

Template DNA
See Template Table below
See Template Table below
BigDye Terminator
(more info)
2 µl
 2 µl*
1 µl
 1 µl*
2.5x Dilution Buffer
(Important buffer info)
(Why 2.5x?)
 2 µl 6 µl* 3 µl 7 µl*
Primer
 4.0 pmol 4.0 pmol* 4.0 pmol 4.0 pmol*
Deionized H2O
 to 10 µl to 20 µl* to 10 µl to 20 µl*
  *For 20 µl reactions increase the volume of dilution buffer and water only.
10 µl reactions are best - use 20 µl only if absolutely necessary.



Analysis Only Template Table

Template
Recommended Quantity
(both 1/4 and 1/8 reactions)
PCR product:  
100 - 200 bp
25 ng
200 - 500 bp
50 ng
500 - 1000 bp
100 ng
1000 - 2000 bp
200 ng
>2000 bp
400 ng
Single-stranded DNA
300 +/- 100 ng
Double-stranded Plasmid DNA
600 +/- 200 ng
BACs, YACs, PACs
0.5 - 1.0 µg + 0.5 µl DMSO
Genomic DNA

2.0 - 3.0 µg + 0.5 µl DMSO

2. Cycle Sequencing Conditions

(Note that temperatures refer to the temperature of the block, not the sample)

3. Remove Unincorporated Dye
by column cleaning using Pharmacia's AutoSeq with G-50 columns packed in H20.

4. Dry Samples Down
in Speed-Vac with no heat.