Sanger Sequencing Protocols
Sequencing supplies for the 3730XL should be purchased by the end user, with the exception of ABI’s Big Dye Terminator (BDT), which may be purchased through JBPC.
You should use the cleanest possible template. Template from the BiomekFX generally works very well for sequencing, and the cost is low. Other acceptable methods of template production are available in kit form from Qiagen, Eppendorf, Millipore, and others. JBPC has a manifold available for the Eppendorf PerfectPrep kit. Contact ABI tech support if you are not certain that your kit will produce sequencing-grade template.
The “largest” reaction used by anyone in the JBPC is around 1/8X, which means a reaction that contains 1/8 of the amount of BDT in the official ABI reaction (1X). The reaction recipes are not simply scaled down in volume. Other modifications have been made. JBPC and most outside users have had good results sequencing clean plasmid templates with these lower reaction volumes. Using smaller reactions saves a substantial amount of money, but is not endorsed by ABI. Also, please remember that what works on one set of templates may not work well on another.
If you need more information about reaction set-up, Applied Biosystems has a good web site (www.appliedbiosystems.com). Particularly useful is the Applied Biosystems 3730/3730XL DNA Analyzers: Sequencing Chemistry Guide: Rev B (part number 4331467B). You can download the guides or buy a hard copy for about $5.00. If you need further help with template concentration, clean-ups, PCR sequencing, sequencing difficult regions, high G-C regions, etc. try ABI tech support. JBPC has 2 protocols for sequencing PCR products; see below. Elena’s protocol has worked very well for her (up to 1 Kbp reads).
Post-reaction clean up and resuspension
Post-reaction clean up is necessary to remove unincorporated fluorescent terminators and other unwanted reagents. Isopropanol precipitations are the preferred choice for simplicity and affordability. More information (and other methods) can be found in the Applied Biosystems 3730/3730XL DNA Analyzers: Sequencing Chemistry Guide (see above).
Cyclesequencing reaction products are fragile, and reactions should be cleaned up by precipitation within 24 hours. After precipitation they should be delivered to JBPC within 3 days. If long reads are a priority, be sure to contact JBPC to schedule your sequencing before you set up reactions.
Name your samples
This is a critical and often overlooked step. The 3730 software has specific requirements for sample names. If your sample names do not follow the guidelines below, we will not run your plate until they are repaired. This is for your own sake. Renaming after a run is possible, but highly time consuming.
Your names should be a simple list of 96 names in a single column in text format.
Do not use spaces or funny characters -@#$?+%^&*)( etc. If any of these characters are used the Data Collection software will not be able to import the sample names.
You can use a period . or an underscore _ to separate information.
The 3730 loads samples in rows of 8 (A1-H1). Your sample names layout should move down, then over. Example A1-H1, A2-H2, A3- H3, etc.
Excel is the easiest way to prepare your sample name list. Save the file in *.txt. (tab delimited text) format.
A handy Excel file for naming samples with simple consecutive numbers can be downloaded here.
Label your plate with your name and plateID. Please write your label on the long sides of the plate, as the short sides will be obscured by plate carriers during sequencing.
The best way to send us your sample names is to attach the TAB-DELIMITED TEXT file to an email to firstname.lastname@example.org. Samples will not be processed without a cost center and default sample names will be used if a sample name file (PLT file) is not provided. Be sure to send it as an ATTACHMENT, not as text within the body of the email.
To bring to JBPC
Your sample plate(s). Plates should be sealed with foil and kept cold and dark.
The JBPC sequencing facility is located in rooms 330/331 on the 3rd floor of the Lillie Building. The Lillie building is located on the corner of Water St and MBL St. The building is locked after hours and on weekends. JBPC sequencing personnel are usually available during regular business hours. You may leave your labeled plate(s) in the freezer. The correct freezer is the one just inside room 330. Samples should be placed on 4th shelf down from the top. If you are new to the process, please check in with a member of the sequencing staff (Leslie or Bette).
Retrieving your data
Shortly after your sequences are finished, you should receive an email from JBPC with a quality assessment and link to the download site. If you are a first time user, we will need to know your lab affiliation to add your email address to the account and to give you a password.