Automated Sequencing Reactions
Part 1. Sequencing Reactions
Terminator BigDye Ready Mix
0.75 µl
5X BigDye Buffer 3.0 µl dH
2
O + Template DNA* 10.25 µl
Primer (3.2 µM)
1.0
 l
Total 15.0 µl
Stir gently to mix ingredients. Keep on ice.
* For purified PCR products, use approximately 20-60 ng DNA; for plasmid preps, use approximately 150-200 ng.
2) Place tubes into a thermal cycler. For PCR products, run program “SEQ40” which has the following parameters:
96° Denature, 1 minute
40 cycles of:
96°, 15 seconds
50°, 10 seconds
60°, 4 minutes
4° Soak
NOTE: For difficult templates, some people find it beneficial to run more than 40 cycles.
I have also found that raising the annealing temp from 50° to 55° can result in cleaner sequences if you think your primer might be binding non-specifically (in more than one place on your template DNA). Longer denaturation steps are recommended for plasmid templates.
3) Following PCR reaction perform the spin column purification protocol listed on the following sheet.
4) Use the Speedvac to dry reactions down to a dry pellet. Set speedvac on medium heat.
5) Add 10 µl of HiDi formamide to the back wall of the tube. Pipet up and down several times, making sure that the formamide contacts the bottom of the tube to resuspend the sample. Transfer sample to 96 well plate. Make sure no bubbles are present at bottom of well when the sample is placed in the 96-well plate. ( If bubbles are present, spin plate in centrifuge equipped to handle plates to remove bubbles.)
NOTE: The 3130xl processes samples in batches of 16. A multiple of 16 wells (16, 32, 48,
64, 80 or 96) must contain samples or blank wells with 10 µl of HiDi formamide.
6) Denature at 95° for 3 minutes in thermocycler prior to loading into the 3130xl.

Part 2 – Clean up reactions
SIUC Fish Genetics Laboratory
Spin Column Protocol
For Cleaning Sequencing Reactions prior to running ABI 3130xl
1) Place the small cap onto the bottom of a Centricon spin column (not really necessary), or just place the column into 2.0 ml collection tube.
2)
Add 700 µl Sephadex G-50 solution to the column. After Sephadex settles a little, examine column to make sure that it contains mostly Sephadex and only a small layer of water above the Sephadex. Remove water and add Sephadex if necessary.
3) If using small cap, remove cap, place into a 2.0 ml tube . Spin at 4000 rpm for 4 minutes. Discard elutant in 2.0 ml tube.
4) Examine columns. Column should have pulled completely away from wall of tube
(see below) but should not contain large cracks.

If column contains large cracks discard.

If fluid is present between the wall of the tube and the column spin at 4000 rpm for 2 minutes and repeat until no fluid remains.
5) Place the finished column into a clean labeled 1.5 ml microtube.
6) Carefully drip the sequencing reaction onto the top of the column. Do not touch the column with the tip of the pipet.
9)
Spin for 4 minutes at 4000 rpm. There should be about 15 µl in bottom of tube.
10) Dry sample using speedvac set on medium heat.

Part 3 – Fill out Sample Sheet for 3130 XL.
1) Open Sequence Sample Sheet template in Excel. “Save as” under a new name for the samples you are planning to run.
2) On the template, the cells that are shaded pink/beige must be filled in. (green are optional)
IMPORTANT: All plate and sample names MUST be formatted with numbers and/or letters and NO spaces. The only special character accepted by the Data Collection software is the underscore symbol: _
Example:
Acceptable: Sample_1
Unacceptable: Sample 1
The same rule applies to the field titled “Container Name”. This field will be the name of your sample sheet when it is imported into the software that runs the ABI 3130xl. Each
“Container Name” must be unique. Sample names do not have to be unique.
The “Owner” name (your name) cannot contain a space.
3) “Sample Name” column:
Make sure every beige-shaded cell contains information.
Files with blank fields will be rejected by the Data Collection software when an “import” is attempted.
Make sure that your sample names correspond to the appropriate wells on your 96-well plate.
NOTE: The 3130xl processes samples in batches of 16. A multiple of 16 wells (16, 32, 48,
64, 80 or 96) must contain samples or blank wells with 10 µl of HiDi formamide.
If you are using less than 96 wells, the sample sheet should list a multiple of 16 samples
(including “NoSample” blank wells if necessary).
Important: Delete rows in the sample sheet that will not be used. Do not simply clear contents. The rows must be deleted to successfully import the sample sheet into the ABI software to run the 3130 genetic analyzer.
4) The cells shaded in green are optional. Do not alter any unshaded cells.
5) IMPORTANT LAST STEP: Saving your Sample Sheet
“Save As” a tab-delimited text file
on a USB drive that you can bring with you when you wish to run your samples. Excel files cannot be imported into the Data Collection software.
Your sample sheet must be in tab-delimited text format.
CAUTION: Any changes to the formatting of the template will likely result in failure to import your sample sheet and will cost time in setting up your sequencing run.