Method: DNA is sequenced in a single PCR reaction containing a DNA
template, appropriate primer, the four dideoxynucleoside teminators, and Taq
FS DNA polymerase. Since the four terminators fluoresce differently, the extended
primers ending in either A, C, G, or T can be distinguished in one reaction
mixture. PCR products are separated by gel electrophoresis and fluorescence
monitored. The fluorescence data is compiled by an ABI 373S DNA sequencer and
the sequence is reported in single letter format and as a four color plot.
Range and Accuracy: Each DNA template yields about 700 bases of sequence
data per primer. After editing the accuracy is about 99% over the first 550
bases. Editing is accomplished by carefully reviewing the four color plot data
and can be performed either by us or by the requisitioner.
Sample preparation: DNA templates will be prepared by the requisitioner. The quality and quantity of the template impacts data quality more than any other single factor. We offer the following recommendations:
| Sample | Purification Method | Concentration | Quantity for one sequencing run |
| dsDNA | Qiagen DNA isolation procedures or Promega Wizard mini-prep | 0.2 µg/µl | 5µl |
| ssDNA | Dynabeads LacZ vector purification kit | 0.1 µg/µl | 5 µl |
| PCR Fragments | Centricon-100 micro-concentrators from Amicon or Perkin-Elmer (P/N N930-2119) | ~10-30ng/µl* | 10 µl |
We recommend E. coli DH5a and HB101 as host strains for template preparation; JM101 should not be used.
Template Quantity: The general recommendations for template and primer concentrations are listed in the chart above. The amount of template needs to be within an appropriate range: if the amount is too low, few complexes form and the signal level is too low to be accurately extracted from the background noise. On the other hand, if the template concentration is too high, the nucleotides in the reaction will be distributed over too many growing chains and an over abundance of short fragments will result.
*A good guideline to follow for PCR-derived template is that 5-10 ng of amplified fragment are required for every 100 bp. Thus only 10-20 ng of a 200bp fragment are needed, and 400ng of a 4kb fragment are needed for an optimal reaction. The reason less template is required for short fragments is that most of the DNA is read, and the concentration of actual template is high relative to the same nanogram amount of a larger fragment.
Sequencing Lamda phage and other large templates: Sequencing of large templates requires very specific concentrations of templates and primers. The template DNA must be at a concentration of 1µg/µl, and at least 5µg should be provided for each sequencing reaction. The primer must be at a concentration of 2.3 pmol/µl, and 10µl should be provided for each sequencing reaction. In order to achieve optimal sequencing of large templates, these concentration guidelines must be followed exactly.
Primers: Custom sequencing primers must be provided by the requisitioner
or purchased at the laboratory at the rates normally charged for DNA
synthesis. Requisitioners should provide at least 10 µl of primer
at a concentration of 0.8 pmol/µl per sequencing reaction. For information
on primer design see section titled General Recommendations
on Primer Design below. Standard primers (M13 forward, M13 reverse,
T7 and SP6) are provided by the laboratory at no cost.
Data: When the analysis is complete the requisitioner will be contacted
via telephone or email. The sequences and the fluorograms can be sent by email
or they can be saved to a zip disk (provided by the requisitioner.) In addition,
we provide the requisitioner with a hardcopy printout of the edited sequences
and fluorograms. The fluorograms are provided as a permanent record of the data
for archiving.
The edited sequence files are in a format ready for downloading to most common sequencing analysis programs. They can also be viewed in MS Word or SimpleText. The fluorograms can be edited and viewed using Applied Biosystems' EditView software (Macintosh only, 1.5 Mbytes) or Chromas software (Windows/DOS.) Free software can be downloaded from Technelysium website ( for PC) or from Applied Biosystems website (for Mac).
Capacity and throughput: With our current system (ABI 373S sequencer and Perkin Elmer 9600 thermocycler), we can sequence 180 templates per week or 9000 per year.
Submitting Samples: Samples for sequencing may be submitted Monday through Friday. The requisition is made on a custom DNA Sequencing Requisition form (PDF). Samples will be sequenced in the order they are received and the data will be returned to the requisitioner less than three days after sample submission.
Sequencing rates :
| # of samples | Price/sample |
| 1-8 samples | $10/sample |
| 9-24 samples | $8.50/sample |
| 25-48 samples | $7.50/sample |
| >49 samples | $5/sample |
General Recommendations on Primer Design: Your
decisions concerning primer sequence, method of primer synthesis, and approach
to primer purification can have a significant affect on the quality of the sequencing
data obtained in Taq DyeDeoxyTM terminator cycle sequencing
reactions. These decisions are particularly important when sequencing is done
on real-time detection systems where signal strength is critical. The purpose
here is to provide you with guidelines to follow in making these important decisions.
Some of these recommendations are based on information that is general knowledge
amongst researchers, while others are based on experience with DyeDeoxyTM terminators.
Information on problems associated with sequencing
Go to the download page to get an order form
(pdf).
The purchase of a multiple peptide synthesizer allows us to offer low cost
peptide synthesis at the following rates:
For peptide synthesis at 25 µmoles
| Peptide Length (# of residues) | Quantity | Delivery (mg) | Cost/AA |
| 5 to 29 amino acids | ~10 to 80 | <5 days | $25 (5 or more peptides, $20) |
For peptide synthesis at 100 µmoles
| Peptide Length (# of residues) | Quantity (mg) | Delivery | Cost/AA |
| 5 to 29AA | >80 | 1 week | $35 |
| 30 to 39 | >160 | 1 week | $45 |
| >40 (on request) |
Peptide purification: $100 per peptide
REQUEST PROCEDURES (choose 1 or 2):
| amino acid | 3-letter code | 1-letter code |
| alanine | Ala | A |
| arginine | Arg | R |
| asparagine | Asn | N |
| aspartic acid | Asp | D |
| cysteine | Cys | C |
| glutamic acid | Glu | E |
| glutamine | Gln | Q |
| glycine | Gly | G |
| histidine | His | H |
| isoleucine | Ile | I |
| leucine | Leu | L |
| lysine | Lys | K |
| methionine | Met | M |
| phenylalanine | Phe | F |
| proline | Pro | P |
| serine | Ser | S |
| threonine | Thr | T |
| tryptophan | Trp | W |
| tyrosine | Tyr | Y |
| valine | Val | V |
Introduction
Mass Spectrometry is a powerful analytical technique for the accurate
mass measurement of synthetic peptides, oligonucleotides, and proteins.
The biopolymers laboratory mass spectrometric analysis uses Matrix-Assisted
Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) instrumentation.
A MALDI-TOF mass spectrometer measures the masses of individual inoized
molecules as a function of their time of flight down a tube of known length.
When performing a Matrix-Assisted Laser Desorption/Ionization (MALDI)
analysis, the sample is embedded in a matrix that enhances inoization of
the sample. The impact of high energy photons from a nitrogen laser pulse
causes the sample to assume a charge, i.e., become ionized.
A time-of-flight (TOF) analyzer is used to determine the masses of samples.
Ions are accelerated by a constant kinetic energy source and directed into
a flight tube of known length. Ionized molecules are separated corresponding
to their time-of-flight through the tube. The velocity of the inoized particles
varies inversely with their masses. The start time is triggered by the laser
pulse and the time measurement is stopped by the impact of ions on the detector.
The detector sends a signal to the digitizer for conversion.
Analytical Services Provided
Synthetic peptides:
Synthetic peptides, glycopeptides, and any modified peptide can be analyzed.
Typical sensitivity is at 1.0 pmol. However, in selected cases the sensitivity
is a few fmol. The mass accuracy is 0.01% with an internal standard and
0.05% with an external standard. Isotopic resolution is achievable up to
about 2 KDa with a mass accuracy of 0.05%.
Proteins:
Proteins can be analyzed up to about 200 KDa with a sensitivity of 1 pmol.
Buffers and salts should be avoided. Typical mass accuracy is approximately
0.05% with external standard and 0.01% with internal standard.
Oligonucleotides:
Molecular weights can be determined up to about a 50-mer with a mass accuracy
of 0.01%. Generally about 5 pmol of material is loaded for analysis. Seqnencing
is possible to provide a 5' to 3' or 3' to 5' sequence of the oligo to verify
sequence or locate modifications on the backbone.
Oligosaccharides:
Sugars can be analyzed at a sensitivity of 5 to 10 pmol with a mass accuracy
similar to that stated above. Typically the mass of the intact glycopeptide
can be measured and then the carbohydrate moiety chemically cleaved and
the mass determined.
Tryptic Digests:
Digests can be analyzed directly from solution at a sensitivity of 1 to
5 pmol. An accurate mass can then be determined and used to search a database
to assist in identifying novel proteins.
Small Molecules:
Small organic molecules (MW <500) can be determined at a sensitivity
of 1 pmol and an accuracy of 0.01%.
Sample Preparation for MALDI-TOF
1. Make a 10 µM solution of the peptide or protein in 0.1% TFA.
2. If salts and detergents cannot be eliminated, try to keep them below the following levels:
The purer your sample, the cleaner, sharper and more intense your spectrum
will be.
Submitting Samples for Mass Analysis
1. Please fill out the Mass Spec Request Form and bring to SGM 330 with sample to be analyzed.
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