The BI sequencing facility ceased operations on 3/1/07

   Official closing announcement, also sent out in Biomail
   Copies of archived sequencing data 1999-2007 are still available. Contact David Mathog for more information.

Local options for sequencing are:



Millard and Muriel Jacobs Genetics and Genomics Laboratory, Caltech.

USC/Norris DNA Core Facility.

Genewiz (San Diego lab)

CIT sequencing service (ends 3/1/07), for comparison, was

There are very many other options for sequencing, but as far as I know all far enough away that FEDEX or other shipping will be required. To find these services google or look through the advertisements in Science and other journals.

For optimum sequencing results, no matter which 3730 machine is used, it is essential that the purified template be at the correct concentration and free of protein, salt and other contaminants. Poor quality template will not sequence well. In the worst cases contaminated template may degrade subsequent runs in the affected capillary.

Data in the above table collected from web sites, emails, and phone calls by David Mathog on 2/21-22/2007.
Last revised 4/07/2007, Added Genewiz 9/28/2011

DNA Sequencing Guide

Updated April 21, 2006.

Quick Overview
Template Type and Quality (miniprep)
   Template preparation
   Template quantity
Template Type and Quality (liquid culture)
Primer design
Primer preparation
Prerequisite SAF account
Submitting samples
Data retention policy and printouts
Retrieving sequencing files: using Netscape (etc.), using Fetch, on Mendel
Viewing traces
Macintosh configuration for 3100/3730 files
Choosing the base calling method and other processing options
BLASTing your sequencing results
Analyzing your data
Sequencer and Instructions Status
Contact Information

Quick Overview

The Core Facility is located in Room 208 of the Beckman Institute. We are currently using the Applied Biosystems 3730 DNA Analyzer. This is a Capillary-Based Sequencer. We now have the ability to sequence from liquid cultures and mini-prepped DNA. We pride ourselves on our turnaround time which is SAME DAY day for mini-prepped DNA and 1 day for liquid cultures. Weekend and Emergency sequencing is available as well.

-Steve Marsh and Tom Lo run the facility

Template Type and Quality (miniprep)

The following templates may be used with the BigDye® terminator chemistry:

Ensuring the quality of DNA in a reaction can affect the performance of the DNA Analyzer. When preparing DNA templates, it is critical to avoid the following:

The presence of residual salts, proteins, RNA, and detergents can interfere with capillary electrophoresis and electrokinetic injection. Your current template purification methods may have to be modified to remove residual salts, proteins, and detergents.

Effect of Residual Salts

Capillary electrophoresis is especially susceptible to salt in samples. The negative ions in salts can be preferentially injected into the capillary array during electrokinetic injection, leading to lower signal. In addition, the negative ions compete and interfere with the injection of larger DNA extension fragments, leading to shortened read lengths.

Effect of Proteins

Many DNA preparation methods for sequencing require the recovery of DNA from lysed bacterial cultures. Unless DNA is carefully purified, protein can remain in the DNA samples. Protein can be injected and adhere to the walls of the capillary array, adversely affecting data resolution and capillary array lifetime.

Effect of Residual Detergents

Some methods of template preparation, such as the Thermomax method for M13 Detergents preparation, use detergents such as Triton X-100 to lyse the protein coat of phage particles. Other detergents, such as sodium dodecyl sulfate (SDS), are used in plasmid purification protocols to lyse bacterial cells. Small, negatively charged detergents may be preferentially injected over DNA during electrokinetic injection. If present at high levels, detergents such as Triton X-100 and SDS will adversely affect the life of the capillary array and the quality of the sequencing data.

Effect of Residual Residual RNA

RNA that is present in DNA template preparations competes with the DNA for injection into the capillary array. Residual RNA has the same effect as excess salt, that is, decreased signal and shortened read lengths.

Template Preparation

Successful sequencing reactions depend upon careful template preparation. Template concentration should be ~60 ng/ul dissolved in water only. No TE or buffer of any kind. We highly recommend using Qiagen or Eppendorf Mini-Prep kits to prepare your templates. When using this kit, DO NOT elute in the Elution Buffer, use water only. If you need to use the elution buffer for larger plasmids please remove the salt from your sample. Perform an ethanol precipitation, rinse the pellet, and resuspend in water. You may use other methods for template preparation at your discretion, but they need to be very clean.  Do not use midi or maxi kits. These kits are too dirty for capillary sequencers.

We need at least 10 uL of template per primer to be used on the template. For example if you want a template sequenced with 3 primers you would need to bring 30 ul of template.

Template preparation for large DNA such as BACS or Fosmids need special consideration. Please call us and we can direct you in the right direction.


Template concentration for sequencing is based on a 5kb plasmid. The molar concentration is what is important. If your sample is not a 5 kb plasmid you should adjust your concentration accordingly.

For example: for a 10kb plasmid double the concentration per lambda.

Please refer to the chart below for your sample application.

Template Quantity

Suspend the following quantity of DNA in 15 uL of water. For example, suspend 900ng of Double Stranded DNA in 15 uL of water to make a solution of 60 ng/uL. Do not exceed the upper concentration!


Recommended Quantity

PCR Product:

    100-200 bp

    200-500 bp

    500-1000 bp

    1000-2000 bp

    >2000 bp

3-9 ng

9-30 ng

15-60 ng

30-120 ng

120-300 ng

Double Stranded DNA

700-1000 ng

Large DNA (BACS, YACS, Cosmids, Fosmids)

1.5 ug-3.0 ug

Bacterial Genomic DNA

6-9 ug

Template Type and Quality (liquid cultures)

The sequencing facility can now sequence from liquid bacterial cultures. This is a very convenient method and saves a lot of mini-prep man hour time. If you would like to use this method you must e-mail or call us by 1:00 PM of the day you are bringing in the samples. Please tell us the number of samples you are bringing. We need to thaw out reagents and this will ensure that we have enough for your samples.

Liquid Bacterial Cultures

Please grow an overnight culture with appropriate antibiotic in LB overnight. Please do not use any media other than LB. Other media can contain inhibitors that will stop the reaction. Target cell ranges are about 10^2-10^4. If you have cell densities that are 10^5 or more, the reaction will most likely fail. Take this culture and dilute it 1:10 to 1:100 in LB in an eppendorf tube. The total amount can be between 100uL to 1 mL. Submit for sequencing.

Notes on liquid cultures:

·        These options are not available for large DNA such as FOSMIDS or BACS.

·        These options will work for circular DNA only. NO linear.

·        You may sequence up to 12 individual reactions for each culture brought in.

·        There is a separate box in the fridge labeled “liquid cultures” for you to submit your samples in.

·        Please make a note on your order form that the samples are cultures.

If you do not call or e-mail us by 1:00PM of the day you are bringing in the sample, your sample may be bumped to the next day.


Primer design

These are the standard sequencing primers we use:

          5'                       3'

We are no longer offering an SP6 primer. There are several different "SP6" sequences and none of these sequence very well. If you supply your own sequencing primers it's very important that these be designed well. To assist you we have provided a web interface to Primer3 as well as an option in tracecontrol which will load your sequences from the sequencing facility directly into the Primer3 page (no cut/paste required.) In both cases the default is to find forward primers, just click on pick primers to perform the analysis. For reverse strand primers uncheck pick left primer and check pick right primer. If you go through the tracecontrol interface you need only select forward or reverse and the appropriate boxes will be checked for you. To test a specific primer paste it into the appropriate box (left or right) and run the program. If no primers are found relax some of the constraints and rerun.

Note that if you check both pick left primer and pick right primer then Primer3 will attempt to find PCR primers which satisfy the size constraints. If what you are really after is forward and reverse sequencing primers run the analysis twice, once in each direction.

Primer preparation

Primers should be a minimum of 21-mers with a concentration of 1-3 pmol/uL in water only, no Tris or other buffers of any kind. See Primer design guidelines. Primer Tm must be greater than 54 degrees C. We need 10 uL of primer per sequencing template. For example if you are using a primer with 3 templates, you would need to bring 30 ul of primer. When bringing templates to be sequenced with multiple primers, please use only 1 tube for the template, not 1 tube for each reaction. Similarly, if one primer is be used with multiple templates give us just one tube of primer holding a volume sufficient for all templates. This saves space for others bringing samples to us.

Prerequisite SAF account

In order to help us keep all of the names and users straight we are requiring that any user of the DNA sequencing facility also have an account with the Sequence Analysis Facility (SAF). If you already have such an account you do not need to apply for another one! This account is free, as is use of the Sequence Analysis Facility to further process your sequences, hint, hint. If you are an on campus user you can apply for a SAF account via the web. Otherwise, send email. In either case, if you have an existing ITS account specify that for the requested username.

Submitting samples

Samples may be dropped off at the Sequencing Facility in room BI 208 from 9:00 AM to 5:00 PM Monday through Friday (except on Institute Holidays). Samples turned in by 12:00 Noon will generally be processed the SAME DAY. Runs are processed in batches of 48 samples and show up on the server approximately every hour (on the bottom of the hour) starting at 6:30 PM the same day.   Please remember that samples are processed on a first come first served basis. If you turn your samples in the afternoon rather than the morning, you may not get your results as quickly as usual. Please ask Steve or Tom if you need a more specific answer on your sample turnaround.

Samples must either be accompanied by a hard copy order form or be submitted after you have filled out the order form on the web!!!
Please fill out the form (paper or web version) carefully as we will not run your sample until all the required information is provided. This includes your PTA account information, PI's name, your Name (first and last), SAF Account, and email address.

Also, please make note of any special conditions your DNA might have. These include but are not limited to:

We guarantee our work, and if you are not satisfied with the results we will rerun the sequence at no charge provided that:

  1. You initially provided us with 10 uL each of template and primer, and the original tubes are still here. If the reactions fail again you will be charged again.
  2. If a special consideration was not indicated, the sample will not be resequenced for free if the condition exists in the template (i.e. G-C rich and we were not notified).

We are on your side. Steve and Tom have over a decade of knowledge of DNA Sequencing that can make life easy for you. If your sample does not work we will try to e-mail you ASAP to discuss your options. Most of the time we can fix the problem with a little more knowledge of your samples.

We are unable to sequence samples for people outside the CalTech/JPL community at this time

Weekends and Emergencies

Data submitted on Friday will be done on the SAME DAY. You do not have to wait until Monday unless you have not turned your samples in on time.  If you have specific questions you should ask either Steve or Tom on Friday.

We understand that there will be times when you will have an emergency and must get data quickly. If you have an emergency we can help you get your data ASAP. Please ask Steve or Tom if you have an emergency.

As soon as you know that you have an emergency please call or e-mail us so we can figure out the logistics. We will try to accommodate your request the best we can.

***If you have more than 20 samples please call ahead to let us know they are coming.

Large Scale Projects

Please come in and talk to Steve or Tom about the particulars of your project. We can compete with other providers and help you gear up your project for the fastest possible turnaround at the lowest possible price.


DNA Sequences $19/rxn

There is no extra charge for GC-rich, BACS, or other difficult templates. However, you must tell us about these problems before we sequence the template!

If you want to bring in 40 or more $10/rxn. Please call ahead. We will instruct you how to bring the samples in.

Data retention policies and printouts

Please be sure to download your sequence files from the ftp or web server and archive them in your lab. The folders in the download area are periodically purged of older files in order to recover space on the server. We keep backups of the data but it is your responsibility to maintain your own data once you have downloaded it. We can usually recover your data from backup media but do not guarantee this service. Printouts will be provided only if you request them. This is because more than 2/3 of the printouts were not being picked up when all samples were printed out. Please indicate in the appropriate field on the request form if you need a printout. If you would like to make a backup CD data disk of your sequences you may bring in a blank CD-R and we will burn it for you. Please call ahead.

You can generally make printouts on your own printer (albeit in slightly different formats) using the PC, Mac, or web based viewers discussed elsewhere.

How to retrieve sequencing files with Netscape (or Mozilla, Firefox, Safari, etc.)

Any version of Netscape will successfully retrieve your DNA sequences. Simply follow the links from the pickup site down to your own files and download them.

We strongly suggest that you if you use a Macintosh that you employ Fetch to retrieve trace files! The .AB1 files produced by the 3100 and 3730 sequencers are stored on the server in a raw binary format.   When downloaded via browser to Windows or Unix these work correctly with the programs on those platforms.   However, most Mac programs need to have both a “file type” and “creator” stamped on the file before they will handle their input files properly.  On Mac OS X you may force this by the following steps:

Download one .ab1 file (hold down mouse on file then select “save target as”).

Select that file on the desktop and do “Get Info”.

Click on “open with” then “other…” and select your application (for instance DNASTAR Seqman or ABI’s EditView).

Click on “change all” to apply this change to all existing and future .ab1 files.

Following this configuration all downloaded .ab1 files will open with the requested application when they are double clicked.  However, they will not open in the application if you click the link in your browser.

How to retrieve sequencing files, using Fetch

Download Fetch 3.0.3 from archive1 or archive2 .  Fetch 4 is commercial and may be purchased from

Since most people will be using Macintoshes, and the FTP program called "Fetch" is widely available as shareware (it is highly likely to be already on the Macs you use), we detail the instructions on how to retrieve your files using Fetch. Before proceeding with the general download instructions please read and out the steps necessary to configure fetch so that the downloaded files will be usable on your Mac.

After opening Fetch, you will go to "File" on the program bar and drag it to "New Connection" which will bring up a window asking to fill in various fields (host, user id, password, and directory). Fill them in as indicated below and click "OK" when finished. (see below)

This will log you on as "anonymous" and take you to the "pub" (short for "public") directory and show you the contents of "pub."

(see below)

There you will find a directory called "dna_pickup". Double clicking that will show a list of directories corresponding to the names of DNA sequencing customers. Find YOUR name and double click it. (see below)

Inside that directory you will find a list of directories with your name AND a date corresponding to the date that the sequencing was completed. This list could go back as far as 2 weeks so note the dates carefully. (see below)

When you find the correct directory of sequences you want, click it twice to view the contents. Go to "edit" and drag it to "select all" or alternatively, press the "apple key" and "a" at the same time to accomplish the same end. All the files you will transfer will be highlighted and you can now click "get files" Make sure the "Automatic" button is clicked (see below)

You will then be asked where in YOUR computer you would like to have your sequences copied to. You can put your files any existing directory, the desktop, floppy, or a new folder (click on New to create it). In the case below, it will be put into "Target Folder" after clicking on "Save." If you forget, you will have to locate the files using "find" in the Finder.

After the "dog" stops running, your files will have been copied onto your local hard drive or floppy, and you are ready to do with them as you wish.

How to retrieve sequencing files, on Mendel

Before you can process your new sequences on Mendel you must copy them from the download area to your personal directory and convert them to GCG format. To do this, start an ssh terminal session on Mendel and issue these commands:

 % setup gcg
 % mydnaget
     This copies all sequence files from your dna_pickup area to a
     subdirectory ~/mydna.  If you have modified some of the files in there
     they will not be overwritten and warnings will appear.
     If the sequence file was in fasta format (output from Phred) it
       will be renamed .nfa.
     If the sequence file was in raw text format (output from the ABI base
       caller it will be reformatted to GCG format and named .seq.
 % myabiget
     This copies all the chromatogram files from your dna_pickup
     area to a subdirectory ~/myabitraces.  If you have modified
     any of the files in there they will not be overwritten and warnings
     will appear.
 % mydnaget copy dna_pickup:[username.newdata]*.seq [].tmp
     Copy the sequence files from the download area to your current
     directroy, giving them a ".tmp" extension to avoid confusing them
     with GCG formatted ".seq" files. 

The ABI trace files are quite large and if you keep many of them in your directory you will soon run out of disk quota. If you want to keep a few around for some reason, copy them to one of your subdirectories, as above. Don't reformat them since they are not simple sequence files. To minimize the disk space that they take up you can compress them temporarily, and then decompress them when you need to use them:

 % gzip mytrace.abi
     Compress the .abi files using GZIP
 % gunzip mytrace.abi.gz
     Decompress a compressed .abi.gz file using GUNZIP.

You may also want to keep copies on some other machine, see the directions for using Fetch and Netscape to learn how to do this.)

Viewing traces

Via the Web

See the section titled: Choosing the base calling method and other processing options

On a Macintosh

ABI has a free program called EditView that you may use to view your trace files and do some crude editing. It only works on OS 9 and below. Here is a location to pick it up.

The site licensed DNASTAR package can view .AB1 files.  Obtain it here:

On Windows

Try Chromas. It can be found at the following site: The latest version is a demo usable for 60 days, an older version (1.45)  is available for free.

The site licensed DNASTAR package can view .AB1 files.  Obtain it here:

Via GCG graphics From Mendel

Move a copy of your trace to your directory on Mendel, and then use the ABIPRINTOUT program to send a copy to your preselected GCG graphics device. Example:

% pdq -h
   This lists all printers, find the one closest to you
   and put that in for "yourprinter" below
% postscript laserwriter "|pdq -P yourprinter"
% abiprintout -infile=trace.abi -begin=100 -end=300
   Prints a region from an ABI trace file to
   the default GCG graphics device.  You can use this
   to review small regions interactively, or to make
   black and white copies through your laserprinter.  The order
   of the traces at each called position are listed top
   to bottom, with the first being capitalized if it is at
   least 5 percent above the next highest base.
% abiview trace.ab1 -outseq=gcg::foo.seq -graph=none
   Extract all bases and writes them into a GCG formatted sequence 
% seqret  foo.seq -outseq=gcg::short.seq -sbegin=100 -send=300
   Extract out bases 100 through 300 inclusive.     
Via X11 graphics From Mendel


If you have an X11 server for your Macintosh or Windows machine you can

use Seqlab, which can display traces.  To start it do:


% seqlab

   Open the trace file from within seqlab.


Macintosh configuration for 3100/3730 files

The newer 3100 and 3730 sequencers are attached to Windows NT machines - earlier ABI sequencers were driven by Macintoshes. One consequence of this is that when the trace files are transferred to the server site they now go as raw binary and have the suffix ".AB1" (the final character is "one", not lower case "L"). Previously they had been MacBinary files and had the suffix ".BIN". MacBinary files contain TYPE and CREATOR information which is attached to the file automatically when it is downloaded onto a Macintosh - this information is not used on any other platform. Unfortunately, the ABI supplied programs Seqed and EditView are too stupid to know how to open an otherwise valid trace file in the absence of this tiny bit of information. In order to force Fetch to stamp this information onto the files as it downloads them you must first have configured the program in the following manner:

  1. Select the Preferences menu
  2. Select Suffix Mapping
  3. Select add
  4. Once in the suffix mapping editor fill in the fields with:

enter suffix=.ab1
name for this type of file = abi 3x00

Once configured in this manner Fetch downloaded files will be accessible via the ABI supplied software.

There may be a way to download these binary files correctly using Netscape or Internet Explorer, but at the moment the only method that we know of requires the use of a tool like resedit to fix up the TYPE and CREATOR information after the fact. Use Fetch instead.

Choosing the base calling method and other processing options

The default base calling method as of January 5, 2003 is once again that provided by the ABI software. However, on the new 3730 it appears that ABI may have switched over to Phred or Phred based algorithms, as limited tests showed no differences in those regions of the trace which were readable.  The 3100 still uses an older base calling method which is definitely not Phred.  In previous years we had been using Phred by default. As with all things, some people were unhappy with one, and others were unhappy with the other. This may not be Burger King, but you can still have it "your way" - at least you can if you're willing to use the web interface which will allow you to call your bases using the local copy of Phred, if you still desire this function. If you do so you will find in addition to your .SEQ files (ABI calls of the sequence) and the .AB1 files (the chromatograms) a set of .NFA files (the Phred calls, NFA = Nucleic FastA). Through this interface you may also perform other simple sorts of trace level analysis, such as selecting the sequence region to display, comparing the trace sequence with a control sequence (look for differences), perform a restriction digest, and annotate subregions of the sequence with simple labels. Here is an example PNG file that shows phred calling, comparison to a very similar sequence, two user supplied annotations, and all enzymes that cut once in the sequence. There is no charge for this service and no username or password is required. Access this service here:

A similar interface is available for carrying out the same analysis for any ABI files that are stored in your SAF directory (not the same as your FTP download directory, which is actually owned by the sequencing facility). In this instance a username/password combination is required (for your protection - you don't want to give the whole world access to your personal files). ABI files obtained long ago or from another source may be processed by first moving them to your home directory (for instance, through W2H using UPLOAD, scp, sftp, or mounting your directory from a Windows client.) Access this service here:

This interface hides the various programs which perform the actual processing, which are Phred, EMBOSS's seqret, GCG's GAP and MAPSORT, and the GCG based ABIPRINTOUT. See here for links to documentation on these programs. You can choose to have the resulting annotated trace sent to the DNA sequencing facility printer, stored as postscript (so that you can print it locally), stored as PNG (so that you can view in your browser), or to not generate any graphics at all. As before, further analysis of your sequence may be performed on the SAF server either in a command line session, or now through the web interface, or X11 interface. To use these extra services you must employ your SAF username/password.

It can take a few minutes for the SAF server to process your sequences once you have initiated this action. If you attempt to download your new sequencing data during the time it is being processed you will likely find either that either the new files you expected (.NFA, .PNG, .PS) are not in your directory yet, or, possibly, you may download part of a file which is still being written. Be aware that browsers cache files, especially image files, so that if you generate a new version of a .PNG file the old one will generally be displayed when you click on that file name a second time. To see the new one force your browser to reload the file from the server. The postscript files are in color, but of course will only print that way on a color printer. The method required to print a Postscript file once downloaded will depend upon the type of computer you use. The most common are:

  1. Macintosh: drag the file onto the color postscript printer icon
  2. Windows: install ghostview and open the file with that, then print from there to your printer.
  3. Unix: lpr

If you have a nonpostscript color printer use Ghostscript or ghostview to send the file to the printer.

If you use Phred to call your sequence the graphic that results will now show a "qual" value under each called base. These are PHRED's estimate of the quality of the call for that base and are equal to:

          q = -10 x log10(p)

where p is the probability of an error for that base. For instance, if p is .01 then q is 20. The larger the value of q the more likely it is that the base has been called correctly. Phred calculates values for p using known information about the behavior of the various chemistries and electrophoretic conditions and the observed chromatogram values. For more information see the following references:

   Brent Ewing, LaDeana Hillier, Michael C. Wendl, and Phil Green.
   Base-calling of automated sequencer traces using phred. I. Accuracy
   assessment. 1998. Genome Research 8:175-185.
   PDF Reprint
   Brent Ewing and Phil Green
   Base-calling of automated sequencer traces using phred. II. Error
   probabilities. 1998. Genome Research 8:186-194.
   PDF Reprint
   Phred Quality Values and ABI 3730 Data

If you want to use the PHRAP assembler on your data you will need to run PHRED manually. For instructions, refer to the UW assembly program section of the on line documentation. Note because of the nature of the license for this software you will need a valid Mendel username/password to read this documentation and/or run this software.

BLASTing your sequencing results

To BLAST your sequence results start here:

Instead of the default processing you may choose to BLAST either the ABI or PHRED base called sequences. The selected sequences may then be submitted to the SAF parallel BLAST server, which will typically be able to process your query faster than the server at the NCBI. This service is also available directly from:

Analyzing your data

There is a very large collection of tools on the Sequence Analysis Facility server which you may use to further analyze your DNA sequences. There is no charge for using these tools is free but you will need to use your SAF username/password to access them through some user interfaces. The GCG and EMBOSS programs are accessible through the W2H web interface (password required). The EMBOSS programs are accessible through the Pise web interface (no password required). If none of the existing tools will perform the required analysis requests for additional programs will generally be honored and/or custom software can often be assembled.   Additionally, site licensed copies of Lasergene’s DNASTAR package may be installed on your Mac or Windows computer.

Sequencer and Instructions Status

In the top level of the dna_pickup folder are two special files:


The first file indicates the status of the two sequencers and the facility as a whole. If one of the machines breaks or some other problem occurs we will notify you through this file. Please check this first before calling us! Click on status to read this file now.

Whenever a change to these instructions is instituted we will update the second file to indicate that such a change has occurred. Please take a moment to review these instructions if you notice that the readme file is more recent than the last time you looked at the instructsions. The date will only be changed for substantial changes (ie, not for corrections to typographical errors and the like.)

Contact Information:

Sequencing Facility: contact for sample problems
personnel: Thomas Lo, Steve Marsh
phone: x2755
room: Beckman Institute 208
hours: 9:00 AM to 5:00 PM Monday through Friday. Closed Institute Holidays.

Sequence Analysis Facility: contact for account problems or for help analyzing your data
personnel: David Mathog
phone: x8453
room: Braun 158
hours: varies