Sample Preparation and Management

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), and 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. Refer to the Applied Biosystems Automated DNA Sequencing Chemistry Guide for more details.

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 (please do not mix). Take care in determining the concentration of your DNA as samples which are too dilute or too concentrated yield poor results.

  Sample Type Min. Quantity Min. Volume Minimum Concentration Preferred Conentration Primer Concentration
Next Generation Seq mRNA-Seq 1.5 μg of Total 25 μL 200ng/μL 200ng/μL  
  Methyl-Seq 100 ng of ChIP'd DNA 10 μL   200ng/μL  
  ChIP-Seq 100 ng of ChIP'd DNA 10 μL   200ng/μL  
  Whole Exome 2-3 μg 10 μL 200ng/μL 200ng/μL  
  Whole Genome 2 μg 10 μL 200ng/μL 200ng/μL  
Standard Sequencing BAC   5-10 μL 1 μg/uL   10 μM
  PCR   5-10 μL 100-500 bp = 6 ng/μL   1.6 μM
      5-10 μL 500-1000 bp = 20 ng/μL    
      5-10 μL > 1000 bp = 30 ng/μL    
  Plasmid   5-10 μL 100 ng/μL   1.6 μM
  siRNA   5-10 μL 200 ng/uL   20.0 μM
  Single Strand DNA   5-10 μL 100 ng/uL   1.6 μM
Genotyping Infinium   25 μL 100 ng/μL    
  Pyrosequencing*   15 μL 50 ng/μL    
Gene Expression Most Arrays   25 μL 60 ng/μL    
  miRNA   15 μL 100 ng/μL    
Methylation     25 μL 100 ng/μL    
DNA Fingerprinting     15μL 50 ng/μL    
*If submitting more than 24 DNA samples for genotyping, please use 96 well plate with cap-based seals. 

 

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), and 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. Refer to the Applied Biosystems Automated DNA Sequencing Chemistry Guide for more details

Note: Templates and primers should only be dissolved in distilled 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.

Plasmids
  • Qiagen
  • Edge BioSystems
  • Promega mini-prep kits***
  • Invitrogen PureLink Quick Miniprep Kit
  • Cesium Chloride centrifugation
  • Modified Alkaline Lysis/PEG method
PCR products with NOnon-specfic products
  • USB PCR Product Pre-Sequencing Kit
  • Qiagen Qiaquick kit
  • Invitrogen PureLink Quick Gel Extraction Kit
  • SAP/EXO I clean-up
  • Exo SAP-IT - USB
PCR products with non-specific products
  • QiaexII Gel Extraction kit
  • Qiaquick Gel Extraction kit
ssDNA (m13)
  • Qiagen Qiaprep M13 kit
  • Thermomax procedure
  • PEG precipitation and phenol extraction
BACs
  • Alkaline Lysis
  • Cesium Chloride
  • Qiagen Large Construct Kit


*** 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. Please refer to the Applied Biosystems Automated Sequencing Chemistry Guide for more details.
  • 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. Or using DNA Mass ladder from Invitrogen (Cat#10068-013).


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).
  • 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:

Primer Sequence
BGH Reverse 5'-TAG AAG GCA CAG TCG AGG-3'
GL primer2 5'-CTT TAT GTT TTT GGC GTC TTC C-3'
M13 Forward 5'-GTA AAA CGA CGG CCA GT-3'
M13 Forward-20 5'-CGT TGT AAA ACG ACG GCC AG-3'
M13 Reverse 5'-CAG GAA ACA GCT ATG AC-3'
M13 Reverse-20 5'-TCA CAC AGG AAA CAG CTA TGA C-3'
pCMV Forward 5'-CGC AAA TGG GCG GTA GGC GTG-3'
pET Forward 5'-TAA TAC GAC TCA CTA TAG GG-3'
pET Reverse 5'-GCT AGT TAT TGC TCA GCG G-3'
pGEX-5 5'-GGG CTG GCA AGC CAC GTT TGG TG-3'
pGEX-3 5'-CCG GGA GCT GCA TGT GTC AGA GG-3'
pMSCV-5 5'-CCC TTG AAC CTC CTC GTT CGA CC-3'
pMSCV-3 5'-GAG ACG TGC TAC TTC CAT TTG TC-3'
pQE Forward 5'-CCC GAA AAG TGC CAC CTG-3'
pQE Reverse 5'-GTT CTG AGG TCA TTA CTG G-3'
RV primer3 5'-CTA GCA AAA TAG GCT GTC CC-3'
RV primer4 5'-GAC GAT AGT CAT GCC CCG CG-3'
SP6 5'-CAT ACG ATT TAG GTG ACA CTA TAG-3'
T3 5'-AAT TAA CCC TCA CTA AAG GG-3'
T7 Promoter 5'-TAA TAC GAC TCA CTA TAG GGG-3'
T7 Terminator 5'-GCT AGT TAT TGC TCA GCG G-3'