Northeastern University

450 Parker St.

Boston, MA 02115

30 May 2017

Dr. Catherine Brownstein

Scientific Director
Boston Childrens Hospital
3 Blackfan Cir
Boston, MA 02115

Dear Dr. Brownstein,

As a current co-op student at the Boston Childrens Hospital Genetics laboratory, I know
firsthand how difficult it can be to transition from book study to performing a laboratory
procedure with high success. Previously, our lab has relied on transfer of knowledge from one
person to the next through repetition to train new co-op students. Although repetition is effective,
it is extremely slow. Instead, requiring Sanger sequencing to be done independently by the co-op
student after initial training completion can speed up the learning process. For this reason, I
created the attached document, A Protocol Guide to DNA Sanger Sequencing. This guide is
meant to facilitate the training of Sanger sequencing for new co-op students by enabling them the
ability to complete the procedure independently.
Dr. Brownstein, I request that you read through the attached protocol guide and pass it on
to the next co-op student for his/her use during the training process. I will also email you a
computer file version of this protocol so that changes can be made as necessary. Ideally, the
document will be amended throughout time, so that it can be passed from student to student with
the latest information.
If you have any questions or comments about the protocol guide, you may reach me at
eric.dahl@childrens.harvard.edu and I will be sure to offer a quick response. Thank you for
considering the implementation of my protocol guide.

Sincerely,

Eric J Dahl
Research Student

Enc. (7)
 A Protocol Guide to DNA Sanger Sequencing
Author: Eric J Dahl
Version 1.0
Updated: 30 May 2017
Contact: eric.dahl@childrens.harvard.edu

Sanger sequencing has been the most widely used method of DNA sequencing since its
inception in 1977. Nearly all of the genetic knowledge that we hold today is due to Sanger
sequencing, with small modifications being made throughout the years to make the process
easier and more reliable. Sanger sequencing is a full day procedure, and it requires extreme
vigilance to ensure high quality results. This protocol guide aims to lay the foundation for
performing DNA Sanger sequencing, and also to foresee any adjustments that will need to be
made based on specific samples submitted.

At the Molecular Genetics Core of Boston Childrens Hospital, we receive DNA sample
submissions from laboratories all over the country. We provide our services to researchers who
do not possess the hardware necessary to sequence samples. Since we provide a service for
compensation, it is necessary to not only be extremely vigilant during the procedure, but also to
help troubleshoot with customers should poor quality results be returned. There are four main
sections to the Sanger sequencing procedure, as outlined throughout this guide. These sections
include Purification, Sample Preparation, Hamilton Cleanup, and Sequencing.

1
 Purification

There are two types of DNA samples that will be submitted for Sanger sequencing: PCR
products and Plasmid samples. Plasmid samples are much easier to work with, as they have
already been purified. If your submission consists entirely of Plasmid samples, purification can
be skipped and you may proceed to Sample Preparation. On the other hand, PCR products must
be purified before continuing.

Materials: ExoSAP-IT PCR Product Cleanup Reagent; vortex genie; mini-centrifuge;

multi-dispense electronic pipette; 96 well plate; 10l multichannel pipette; plastic seal;
centrifuge; thermocycler

Procedure:
1. Vortex and centrifuge the ExoSAP-IT reagent
2. Set the multi-dispense pipette to 2l, and uptake ExoSAP-IT reagent
3. Dispense 2l of ExoSAP-IT into a 96 well plate for each PCR product sample that has
been submitted
4. Set the multichannel pipette to 5l, and transfer samples from their original housing to
the 96 well plate with ExoSAP-IT already added
5. Seal the 96 well plate with a plastic wrap
6. Vortex and centrifuge the 96 well plate
7. Place the plate in a thermocycler, and run the exosap005 option

After the samples are placed in the thermocycler, they will be purified in approximately
5-10 minutes. When the thermocycler shows the symbol for time left during step, this means
the samples are ready to be taken out and sample preparation can begin.

A mini-centrifuge (left) and vortex genie (right), as used in step 1.

2
 Sample Preparation

Now that all samples are purified, the next few steps are to add primers and cocktails
together with the samples in a 96 well plate before putting them back into the thermocycler.
Before handling the samples, it is smart to have a plan. Researchers submit their samples with
sample sheets, and these sample sheets should be marked with each sample corresponding to the
intended well location in the 96 well plate. After the intended plate layout is planned, it is time to
start adding the cocktails and primers, both of which are already prepared. Again, Plasmid
samples will be easier to work with because they do not require the addition of a primer while
PCR products do. Primers are submitted separately by the researchers. Lastly, a sample named
PGEM is always added to the plate as the control sample.

Materials: Plasmid cocktail; PCR cocktail; PGEM; vortex genie; mini-centrifuge; multi-
dispense electronic pipette; 96 well plate; 10l multichannel pipette; 10l single channel pipette;
plastic seal; centrifuge; thermocycler

Procedure:
1. Make a plan about the wells in which you intend to add each sample, and keep track
whether they are a Plasmid sample or a PCR product
2. Vortex and centrifuge all purified samples, cocktails, primers, PGEM
3. Set the multi-dispense electronic pipette to 4l
4. Add 4l of Plasmid cocktail and/or PCR cocktail to the plate as necessary (PGEM
requires a Plasmid cocktail)
5. For Plasmid samples only, set the multichannel pipette to 6l and transfer the samples to
the 96 well plate with Plasmid cocktails already added (Including PGEM)
6. For PCR products only, set the multichannel pipette to 5l and transfer the samples to the
96 well plate with PCR cocktails already added
7. For PCR products only, use the single channel pipette to add 1l of primer to its
respective PCR product sample
8. Seal the 96 well plate with a plastic wrap
9. Vortex and centrifuge the 96 well plate
10. Place the 96 well plate in a thermocycler, and run the SEQUENCE2M option

3
 A thermocycler, as used in step 10.

After placed in the thermocycler, samples will be ready for Hamilton Cleanup in
approximately 2 hours. Use this time to prepare the Hamilton workstation.

Hamilton Preparation
1. Add the Hamilton pipette racks to their proper positions inside the Hamilton workstation
2. Add 85% ethanol and distilled water to their respective reservoirs inside the Hamilton
workstation

When the thermocycler has completed its countdown for Time Remaining, the samples are
OK to take out and move on to Hamilton Cleanup.

4
 Hamilton Cleanup

After the samples have been taken out of the thermocycler, the remaining sections of the
protocol are relatively straightforward and mostly handled by machines. After a manual addition
of Cleanseq beads to the samples, the 96 well plate is placed inside the Hamilton workstation,
where the Hamilton will automatically clean up the samples. This process is needed to remove as
many impurities as possible for a high quality reading by the DNA analyzer.

Materials: Cleanseq beads; 10l multichannel pipette

Procedure:
1. Set the multichannel pipette to 10l, and add 10l of Cleanseq beads to each sample well
2. Place the 96 well plate inside the Hamilton workstation
3. Open the Nimbus program on the connected laptop, and start the DNA Cleanup
procedure
4. Follow any manual steps from the on screen prompts

The Hamilton robot workstation will take the procedure from there. There are some
manual steps that will be prompted on the computer screen, so it is important to check back on
the workstation often. After about 40 minutes, the computer will show a green check mark
indicating procedure completion, and the plate will be ready for DNA sequencing.

The Hamilton work station.

5
 Sequencing

Samples have been purified, mixed with appropriate cocktails & primers, and impurities
have been removed. It is now time to sequence the DNA.

Materials: DNA analyzer casing; rubber topper

Procedure:
1. Take the 96 well plate from the Hamilton workstation and place a rubber topper on top
2. Place the 96 well plate with rubber topper in a DNA analyzer casing
3. Place the casing inside of the DNA analyzer
4. Click start in the DNA analyzer program on the connected computer

All manual steps of the protocol are now complete. The DNA analyzer will take about 4
hours to sequence all samples in the 96 well plate, and analysis completion will be indicated on
the computer screen.

The 3730 DNA analyzer, also known as the sequencing machine.

6
 DNA Analysis & Closing Notes

Once the sequencing run is complete, the sample files will be located in the Results
(seq) folder on the computer. These files can be opened with the Sequencing Analysis v6.0
software. When the files are opened, distinct base reads should be visible, along with signal
strength. An example file is shown below.

An example DNA analysis file.

The information of most interest in this file is the signal strength, as indicated by the blue
bars at the top of the screenshot. Blue bars indicate excellent signal strength, yellow bars indicate
satisfactory signal strength, and red bars indicate poor signal strength. If there are multiple bases
with poor signal strength, it is necessary to troubleshoot with the customer to find the culprit of
the poor results. There is a 3730 Troubleshooting Guide in PDF format on the computer that
can be utilized to narrow down the cause. In the case that the culprit has been found to be at the
fault of the Molecular Genetics Core of Boston Childrens Hospital, it is advised that a free rerun
of samples is offered to the researcher as retribution.

This DNA analysis concludes the protocol guide to DNA Sanger sequencing. If there are
comments or questions regarding the use of this protocol guide, please do not hesitate to contact
the author, Eric J Dahl, at eric.dahl@childrens.harvard.edu and a prompt response will be
offered. Good luck.