DNA Sequencer
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:
- Ben tenOever (tenOever [-at-] mcb [dot] harvard [dot] edu, Fairchild 489)
- Sean Buchanan (buchanan [-at-] mcb [dot] harvard [dot] edu, Fairchild 493 )
- Stefanie Schalm (schalm [-at-] fas [dot] harvard [dot] edu, Fairchild 481)
Any of these operators can be contacted for information regarding staff-run sequencing.
Sequencing Trainer:
- Christian Daly (cdaly [-at-] cgr [dot] harvard [dot] 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 webmaster [-at-] mcb [dot] harvard [dot] edu.
For general questions about the facility, contact Claire Reardon (claire [-at-] cgr [dot] harvard [dot] edu).
Domain Accounts
All Sequencing Facility users must have an MCB or CGR 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 here. Requires a Harvard PIN.
Sequencing Account and Slot Sign-Up
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 new codes to your existing account, please contact Jennifer Xie in the MCB financial office (jxie [-at-] mcb [dot] harvard [dot] 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 four 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:
| 8.0 uL | BigDye Terminator Ready Reaction Mix |
| 3.2 pmoles | Sequencing Primer |
| 15-30 fmoles | DNA template |
| 20uL | total reaction volume |
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 [-at-] lsdiv [dot] harvard [dot] 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
| 24 Ready Reactions | P/N 4337454 |
| 100 Ready Reactions | P/N 4337455 |
| 1,000 Ready Reactions | P/N 4337456 |
| 5,000 Ready Reactions | P/N 4337457 |
| 25,000 Ready Reactions | P/N 4337458 |
The Sequencing Facility sells 100-reaction aliquots for $610. To purchase an aliquot, please fill out this form and email it to 3100user [-at-] mcb [dot] harvard [dot] edu. You can pick up your aliquot from the sequencing staff.
MicroAmp® 8-Strip Reaction Tubes
| 1,000 Tubes; 125 Strips | P/N 801-0580 |
ABI PRISM™ Optical Caps, 8 Caps/Strip
| 300 strips/pkg; 2,400 caps/pkg | P/N 4323032 |
Princeton Separations (http://www.prinsep.com)
| Centrisep Spin Columns | Catalog # CS-900/CS-901 |
| Centrisep Spin Sep-8 | Catalog # CS-912 |
Staff-Run Samples
Users must have an MCB or CGR Domain Account and must register for a Sequencing Account. Please use these instructions to obtain an account if you do not already have one.
Reservations and Costs
All sample slots must be reserved on the web signup page prior to submission. Samples that haven’t been scheduled will not be loaded.
Reservations are accepted in increments of 8 samples, also referred to as a slot (1 slot = 8 samples). The facility can handle 144 slots per day. The fee per slot is $16; this will be charged to the 33-digit code that you supply when you reserve the slot. We offer a discounted price of $96 for full plates (96 samples = 12 slots). This discount will be applied automatically when you reserve 12 slots at one time.
Sample Labeling and Submission
Samples may be submitted in 0.2ml tubes or plates. Please use a plate that is compatible with the sequencer (for example, VWR part #83009-676). The samples can be capped, parafilmed or scotch taped to prevent evaporation and spills. Taped or parafilmed tubes are preferred. We do not suggest wrapping the tubes into aluminum foil as it is a problem to unwrap them. The length of exposure to light will not significantly diminish the signal.
Each submitted strip tube or plate must be clearly labeled so that the order of samples and domain account name of the user are legible. We recommend the user labeling strip tubes as indicated below using an indelible smudge-free pen, such as VWR Lab Marker. If the full name of your account cannot be written, do not worry, as we will be able to recognize user account name by looking at your user profile from the web signup. We do not need any other information - everything else is available in your user profile. Please always label the tubes and not the caps. The caps will be removed upon placement in the machine and the proper orientation can be lost.
Samples must be dropped off at the Sequencing Facility before 5pm of the loading day.
Data Retrieval
Sequence data from each sample are in a proprietary-format file containing a four-color electropherogram. The files also contain various information regarding the strength of the signal, sequence data in text format, etc. Once the samples have been analyzed, the resulting data files are transferred to the user’s folder.
Usually, the sequenced data will be put in your folder within 17 hours. This folder is only a temporary storage place – please move the data to your own permanent storage within 24 hours of receiving it, and then erase it from your folder. Data left in users' folders longer than a week may be removed without backup or warning.
Getting your files from the server:
This method can be used from a computer in Biolabs, Fairchild, or Bauer
PC:
On your desktop, right-click "My Computer"
Click on Tools, Map Network Drive
Accept whatever drive letter appears
In the folder field, type: \\titan2\dna or \\titan2.mcb.harvard.edu\dna
Scroll for your folder and click <Finish>
Your folder should open on your desktop.
Drag your files from this folder to your local drive
(it’s wise also to make a backup onto other media).
Verify that the local copies of the files are readable.
Drag the files on the server to the recycle bin and empty it.
Mac:
This only works for OS X 10.3.9 and above.
Hit Apple-K, then enter: smb://titan2.mcb.harvard.edu/dna
Scroll for your folder.
A server volume icon with Microsoft’s logo should appear on your desktop.
Double-click to open it, and drag your sequencing files to your local drive
(it’s wise also to make a backup onto other media).
Verify that the local copies of the files are readable
Drag the files on the server to the trash and empty trash.
Getting your files from the ftp site:
This method can be used from any computer with an internet connection.
You will need to use your CGR or MCB username and password to authenticate.
Point your web browser to ftp://www.mcb.harvard.edu/dna
Scroll for your folder.
Drag your files from this folder to your local computer.
(it’s wise also to make a backup onto other media).
Self-Run Samples
Each lab in Life Sciences may designate one member to become a “power user” of the Sequencing Facility. Power users are trained by our staff to independently run their plates on our sequencers. For information about our instruments, please see Equipment.
To schedule a training session, please contact Christian Daly (cdaly [-at-] cgr [dot] harvard [dot] edu). We charge a training fee of $220 per session. We will ask you for a 33-digit code that will be used to charge both the training fee and your subsequent usage fees (two different codes may be used). Please ask your PI or lab administrator for the appropriate code.
Once trained, power users are given an account that allows them to log in to the instruments. Users must also register for a Sequencing Account on the web signup page in order to be able to reserve time on the instruments. We allow reservations in one-hour time blocks. A typical run takes two hours, but longer runs may take up to four. You must also record how many plates you will run during your scheduled time so that we can correctly charge your account. You may cancel a reservation up to two hours before the scheduled start time. After that, the scheduled run will be charged to your account.
We charge a fee per plate to recover the cost of reagents and instrument maintenance; $100/plate for runs on the 3730xl sequencers or $75/plate for runs on the 3130xl sequencers. You cannot run partial plates as a power user. If you have fewer than 96 samples, consider submitting them to be run by our staff.
Once your samples have run, please come back to retrieve your data. You may transfer your data using our temporary server space on \\titan2 or with a USB key. To save to \\titan2, go to Start, Run, and type \\titan2\dna. Open your folder and transfer your data there. You can now retrieve your data from any computer with an internet connection. You will need to use your CGR or MCB username and password to authenticate. Point your web browser to ftp://www.mcb.harvard.edu/dna. Scroll for your folder. Drag your files from this folder to your local computer (it’s wise also to make a backup onto other media). Once you have confirmed the successful transfer of your data, please delete the original off the hard drive. Any data older than one week is subject to deletion without warning.