Sample Preparation and Management

The quality of your sequencing template greatly affects the success of the sequencing reaction. Sequencing by capillary electrophoresis is highly sensitive to sample contamination. Potential contaminants include: proteins, RNA, chromosomal DNA, non-specific PCR products, residual salts, organic chemicals (i.e. phenol, chloroform or ethanol), residual detergents, as well as excess PCR primers, NTPs, enzyme and buffer components from PCR reactions. Poor quality DNA can cause noisy data (i.e. peaks under peaks), unusable sequence data, or weak signal in addition to diminishing the lifetime of the capillary array.

The following preparation methods have been shown to give reliable data when protocols are followed carefully. 

Concentrations

All samples and primers must be dissolved in distilled water and must be provided at the concentrations below. For each sequencing reaction submit 10 μL of template and 10 μL of primer (templates and primers can be mixed or not mixed). Take care in determining the concentration of your DNA as samples which are too dilute or too concentrated yield poor results.

Sample Prep for Sanger Sequencing

Template Preparation

The quality of your template greatly affects the success of the sequencing reaction. Sequencing by capillary electrophoresis is highly sensitive to sample contamination. Potential contaminants include: proteins, RNA, chromosomal DNA, non-specific PCR products, residual salts, organic chemicals (i.e. phenol, chloroform or ethanol), residual detergents, as well as excess PCR primers, NTPs, enzyme and buffer components from PCR reactions. Poor quality DNA can cause noisy data (i.e. peaks under peaks), unusable sequence data, or weak signal in addition to diminishing the lifetime of the capillary array.

The following DNA template preparation methods have been shown to give reliable data when protocols are followed carefully. 

Note: Templates and primers should only be dissolved in MilliQ (18ohm) water! The addition of any additional EDTA or divalent cations will negatively affect your sequencing reaction.

Please note: Other preparation methods and kits exist and these are only suggestions.

*** We have observed that DNA samples purified using spin columns often contain residual spin column resin which leads to poor sequencing results. We recommend an additional centrifugation step following spin column elution. We have found that centrifuging the eluted sample for 5 minutes at maximum speed and removing only the uppermost portion of the sample to a fresh tube avoids the carryover of the interfering column resin.

DNA Quality Determination

The following methods should be used to examine your DNA quality:

Agarose gel electrophoresis
Agarose gels indicate the presence of non-specific PCR products and RNAs. If you find non-specific PCR products, you should use one of the agarose gel purification method listed above. 

Spectrophotometry
Spectrophotometry indicates the presence of protein contaminants. For spectrophotometry, the ratio of A260/A280 ration should be 1.7-1.9. Smaller ratios usually indicate the presence of proteins or organic chemicals. Ratios greater than 1.9 indicate the presence of contaminating RNA.

DNA Quantitation

DNA quantity is very important to the success of your sequencing reactions. Three common ways to determine DNA quantity are spectrophotometry, fluorometry, and agarose gel electrophoresis.

Spectrophotometry

In spectrophotometry you measure the absorbance (optical density or O.D.) of a sample at 260 nm.

Fluorometry

In fluorometry you measure the fluorescence of the dye bisBENZIMIDE (Hoechst 33258) binding to AT sequences in the minor groove of DS-DNA. This method is specific for quantitation of nanogram amounts of DNA. (See Sigma technical bulletin # MB-590; Product # DNAQF, or Picogreen from Invitrogen Cat #P7589).

Agarose Gel

If you plan on quantitating with an agarose gel, we strongly advise using a mass ladder (VWR Scientific PN 80509-748), the mass ladder will allow a quick relatively accurate quantitation. 

Primers: 

• Your decisions concerning primer sequence, method of primer synthesis, and approach to primer purification can have a significant effect on the quality of the sequencing data. Some of these recommendations are based on information that is general knowledge amongst researchers, while others are based on experience with dideoxy terminators.
• Not all cloning vectors are created equally. Several versions of common sequencing primers exist and DO NOT hybridize well with all vectors. Check to be sure that your primer has exact homology to your cloning vector of choice.
• Primers should be at least 18-20 nucleotides in length to minimize the chances of encountering problems with a secondary hybridization site on the vector or insert.
• Primers with long runs of a single base should generally be avoided. It is especially important to avoid 3 or more G's or C's in a row.
• For cycle sequencing, primers with melting temperatures above 50°C generally produce better results than primers with lower melting temperatures.
• Primers should have a G/C content between 40 and 60 percent. For primers with a G/C content of less than 50%, it may be necessary to extend the primer sequence beyond 18 bases to keep the melting temperature above the recommended lower limit of 50°C.
• Primers should have a GC clamp on the 3' end.
• Primers should not contain palindromes. Palindromes can cause hairpins loops within the primer, thus resulting in an unproductive priming event.
• Primers should not contain sequences of nucleotides that would allow one primer molecule to anneal to itself or to the other primer used in the sequencing reactions (primer dimer formation).
• When possible run a computer search against the vector and insert DNA sequence to verify that the primer is unique.
• Do not design degenerate primers. Do not request inosine in sequencing primers. Both degenerate primers and primers containing inosine often either fail during sequencing or give poor results.

We provide some free standard primers for sequencing. Please check their sequences below before you use them: