Available Services:

Sequencing Facility services are available to the members of the Life Sciences Division at Harvard University which includes the MCB, OEB, CCB, Biological Anthropolgy, and Psychology departments.  We offer the following sequencing options:

1.  Staff-run analysis of dye terminator sequencing reactions.

Drop off your reactions ready to run on the sequencer.  The staff will run them and put your data on the central server about 17 hours. 

 2.  Self-run use of our 4 sequencers.

After training by our staff, you will be given “power-user” status and may run your own plates on our sequencers.  Only one “power user” per lab, please.

Location:

The Sequencing Facility is located in Biolabs 3094. A small shelf next to the door is provided for submitting the samples.

Contacts:

Current Operators:

• Sean Buchanan (buchanan@mcb.harvard.edu, Fairchild 493 )
• Hon-Ren Huang (hrhuang@mcb.harvard.edu, Fairchild 493)

Any of these operators can be contacted for information regarding staff-run sequencing.

Sequencing Trainer:

• Christian Daly (cdaly@cgr.harvard.edu)

Contact Christian if you would like to be trained to run your own plates on the sequencer.

For questions regarding the sequencing web signup page, contact the . 

For general questions about the facility, contact Claire Reardon (claire@cgr.harvard.edu). 

Sequencing Account and Slot Sign-Up

All Sequencing Facility users must have an LSDIV Domain Account in order to use the signup and scheduling pages and to be able to retrieve sequencing data. If you don't have an MCB or CGR Domain Account, apply for one through the link below.

Ask your Lab Administrator or PI for the appropriate 33-digit billing code(s); you'll need at least one to set up your Sequencing Account and to reserve sequencing slots.  To add codes to your account, please contact Jennifer Xie in the MCB financial office (jxie@mcb.harvard.edu). 

Sequencing Account sign-up and slot reservations are available here.  Users outside of MCB must use the log-in version in order to access the sign-up page.
The first time you use this page you will be asked to supply your name, phone number, email address and lab. Enter the correct information and click "Submit Info." This creates your Sequencing Account.

Proceed to the appropriate calendar. After you've selected a date, you will be asked for up to two 33-digit billing codes to which your run will be charged. The codes that appear in the pull-down menu will only be those for your lab (use the Code Helper to create descriptive labels for your lab's codes). After you've selected the appropriate code(s), your reservation is complete.

Sample Preparation:

Template Preparation
We recommend Qiagen miniprep kit for sequencing plasmid DNA and Qiagen PCR purification kit for sequencing PCR products.

Setting up Sequencing PCR reaction
Standard reaction contains the following:

The standard reaction can be halved or quartered to economize the BigDye Terminator Ready Reaction Mix.

PCR Cycle Sequencing
25 cycles of
   96°C 10 seconds
   50°C 5 seconds
   60°C 4 minutes
hold at 4°C (or store at -20°C) until ready to purify the reaction samples.

Sample Purification
Column purification of the PCR sequencing samples is strongly recommended. We recommend Centrisep columns from Princeton Separation, but there are other similar products, that should work as well.

Sample eluate from the column must be transferred to 8-strip tubes (0.2uL tubes) or plates (for example, VWR part #83009-676).

The minimal volume that can be handled by the machine is 10uL. The recommended final volume for the sample is 20uL. Therefore, we suggest addition of 10uL water to the sequencing reaction volume before column purification if your sequencing reaction was done in 10ul.

Equipment:

The facility has two 96 capillary ABI3730xl Genetic Analyzers and two 16 capillary ABI3130xl Genetic Analyzers.  These instruments are configured to analyze samples produced by thermal cycle sequencing with AmpliTaq DNA Polymerase FS, and fluorescent dye-labeled dideoxy-terminators (BigDye Terminators). Detailed protocols on template preparation, PCR reaction mix preparation, cycling conditions, and post-PCR purification are available at http://www.appliedbiosystems.com.  All of our instruments can also run genotyping and fragment analysis using the GeneMapper software.

A GeneAmp 2400 thermal cycler (available in Fairchild 491) is programmed with a method described by Applied Biosystems for the BigDye-terminator chemistry in a 0.2-ml MicroAmp PCR tube. Please contact the operators if cycling parameters (e.g. annealing temperature) must be different than those set in the programmed method.

Data Analysis:

MCB licenses VectorNTI for Windows and MacOS, and strongly encourages MCB department members to use it.  You are welcome to use software other than VectorNTI, but if you do so, you will have to provide your own support and troubleshooting.

Instructions for downloading VectorNTI software for your PC or Mac are here (viewable only within MCB).

If you have questions about data transfer, networking, or software downloads, contact support@lsdiv.harvard.edu.

Core Reagents:

Following is a list of reagents mentioned in this document for your convenience.

Applied Biosystems (http://www.appliedbiosystems.com)

ABI PRISM® BigDye™ Terminator v 3.1 Ready Reaction Cycle Sequencing Kits with AmpliTaq® DNA Polymerase, FS

The Sequencing Facility sells 100-reaction aliquots for $610. To purchase an aliquot, please fill out this form and email it to 3100user@mcb.harvard.edu. You can pick up your aliquot from the sequencing staff.

MicroAmp® 8-Strip Reaction Tubes

ABI PRISM™ Optical Caps, 8 Caps/Strip

Princeton Separations (http://www.prinsep.com)