sanger SEQUENCING

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DO:

• WRITE CLEARLY
• USE SHORT, DISTINCT NAMES FOR ALL SAMPLES
• INDICATE IF YOUR PRIMER IS ALREADY ADDED
• INDICATE IF YOU WOULD LIKE (OR NEED) THE dGTP PROTOCOL (SEE BELOW)
• INCLUDE THE PRINTED FORM ALONG WITH YOUR SAMPLES
• INCLUDE CUSTOM PRIMERS WITH THEIR CONCENTRATION AND CLEAR LABELING
• USE BUBBLE WRAP OR STRONG SECONDARY CONTAINERS FOR SHIPPING (50mL TUBES, ETC). 
• USE UPDATED FORMS IF POSSIBLE
• REACH OUT TO US IF YOU HAVE ANY QUESTIONS BEFORE SEQUENCING

DO NOT:

• USE PARAFILM
• FORGET THE ORDERING FORM
• FORGET TO UPDATE YOUR INFORMATION AT THE TOP OF THE ORDER FORM(S)
• SEND SAMPLES ON DRY ICE (THEY CAN IN FACT BE SHIPPED AT ROOM TEMPERATURE)

Please follow these guidelines when preparing your samples:

Each sample tube (1.5ml tubes preferred) should contain one primer and one template at the quantities specified below:

• ssDNA plasmid:  0.25μg DNA/5kb    + 2 pmol primer in 13 µl
• dsDNA plasmid:  0.5μg DNA/5kb      + 8 pmol primer in 13 µl
• PCR products:    2-4ng DNA/100bp  + 8 pmol primer in 13 µl

Please note that the values listed for the primer are quantities, not concentrations.

A simple and approximate usage is to include (into the total 13µL volume), either: 

• 1µL of 10µM primer, or 
• 3.2µL of 3µM primer 

If requesting one of our stock primers, put a large note in red on your order form, and submit samples in 10uL total volume.

Template: Please be sure that you do not have SALTS, PROTEIN, DETERGENTS, RNA, CHROMOSOMAL DNA, ETHANOL, or EDTA.

Quantify your products on an agarose gel or use a NanoDrop. Please let us know if your products are at a low concentration and/or are larger than 10kb.

Primers: Our default annealing temperature is 50°C.  Please let us know if this is not compatible with your primer(s), and please indicate the required temperature. 50°C is preferable if possible.

Order forms can be filled out and printed from this web site by following the DNA Sequencing Order Form Download Link

You may also submit samples in a 96-well plate. We charge per sample, so you don't pay for empty wells. Please keep well A1 empty for our positive control.

We prefer samples to be submitted in a specific type of plate:

• Catalog#: 4ti-0770/c
• Description: FrameStar 96, semi-skirted
• To order, please contact Dennis Harte @ 510-882-0640 or www.BasicScientific.com
• You may purchase these plates at the DNA Sequencing Facility for $6 each
• If you are shipping plates, please use very tight strip caps.

When submitting samples in a plate, the total volume per well should be 6µL that includes:

dsDNA plasmid: 250ng/5kb  + 4 pmol primer or PCR product: 20ng/1000 bp  + 4 pmol primer.

If you are requesting the dGTP protocol due to the presumptive presence of secondary structure in your target sequence, please reduce the volume of your template+primer to 4µL.

Please download the Full Plate Sequencing Order Form, e-mail us a copy and print a copy to include with your plate. 

When filling out the form, please keep the following in mind:

• Sample names cannot have spaces, but may include the following:  -+.(){}_#
• Sample names may be up to 35 characters long.
• Indicate a unique plate name or barcode on the order form, and write it on the side of the plate

For templates larger than 10KB, please provide 1µg of template with 10pmol of primer in total volume of 13µL.

In addition, please write "Large  Template Protocol" in large print across the order form.

IMPORTANTLY -- If you suspect that there may be a secondary strucure in your template, please indicate in BIG RED MARKER that you want "dGTP PROTOCOL"

dGTP protocol may also work with GC and GT-rich templates.

We are testing a new sequencing protocol for cosmid, BACs, PACs, and Fosmids.  We will sequence your sample for free of charge. If you have a  protocol you want us try, we will be happy to try it.

Please provide:

• 5µg template with 30pmol of primer in total volume of 12µL.
• Write the Tm of your sequencing primer on the order form.

Things to consider when preparing Cosmid, BACs, PACs and Fosmid for sequencing:

• Template quality:  Avoid genomic DNA contamination by not allowing cells to remain in the lysis solution longer than the recommended time in the protocol.  Do not votex nor freeze your DNA.  Freshly prepped DNA gives better results.
• Recommended kits: Qiagen Genomic-tip or Promega Wizard Genomic Kit

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