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Sanger-based DNA Sequencing

The SMF provides DNA Sequencing from single stranded or double stranded DNA, from purified plasmids, PCR products, and BACs. Sequencing is performed primarily on ABI 3730XL and 3730 DNA sequencers using Big Dye terminator cycle sequencing chemistry. The facility quantifies all samples, performs sequencing reactions, cleanup and capillary electrophoresis, analyzes the data and provides sequence as text files and chromatograms.

Turnaround Time: 24-48hrs (weekdays)

Longer turnaround times may occur when our sample volume is very high , when special conditions are requested and when we experience instrument problems.

Guidelines for DNA Submission

Sample Submission Requirements
Plasmid concentration must be 100 ng/µl. Submit 10 µl per reaction . Custom primers must be at 1 pmol/µl. Submit 10 µl per reaction. The DNA and primer must be submitted in 0.5-ml Eppendorf tubes with the sample name written on the sides and tops of the tubes. BAC DNA should be submitted at 500 ng/µl and primers at 25 pmol/µl. PCR products should be submitted at a minimum concentration of 20 ng/µl for products less than 1 kb. Products 1 kb or greater should be submitted at 30 ng/µl.  Submit 10 µl per reaction

Quantitation of DNA
All DNA submitted to the facility needs to be accurately quantified. We recommend visual determination of DNA quality and quantity on an agarose gel using a quantitative DNA ladder. Alternatively DNA concentration can be determined fluorometrically. DNA concentrations determined using a spectrophotometer are often artificially high due to the presence of RNA, proteins, bacterial genomic DNA and other contaminants.

Note: Low DNA concentration is the most common cause of poor quality sequence and failed reactions. Too much DNA can, however, be as bad as too little. The presence of too much template results in top-heavy data (strong peaks at the beginning which fade rapidly), pull-up peaks (non-specific peaks that appear below the correct peak) and loss of peak resolution. In addition, it shortens the life of our capillaries.

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Custom Sequencing Primer Design Guidelines

  1. Thermal cycling Conditions: Denaturing step heats the reaction mix to 96°C. The annealing step cools the reaction mix to 55°C, and the polymerization step extends at 60°C. Primers must have annealing temperatures of 56°C or higher.
  2. Primer length should be at least 20- to 25-mers. GC content should be 50% or more.
  3. Primers should be designed with a tightly binding 3' end.
  4. When designing a primer, do not pick a region that is closer than 50 bases to the region of interest.
  5. Primers for PCR reactions tend to work fine for automated sequencing.

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Sequencing Primers Provided by the Facility

The facility currently provides the following primers free of charge.

T3
T7
T3-0
T7-0
M13(-21)
M13rev
Sp6
Bluescript KS
Bluescript SK
BGH Rev
PCMV For
pGEX 3'
pGEX 5'
T7 Term
CCT CAC TAA AGG GAA CAA AAG C
TAA TAC GAC TCA CTA TAG GGC GA
ATT AAC CCT CAC TAA AGG GA
TAA TAC GAC TCA CTA TAG GG
GTA AAA CGA CGG CCA G
TCA CAC AGG AAA CAG CTA TGA C
GAT TTA GGT GAC ACT ATA G
TCG AGG TCG ACG GTA TC
CGC TCT AGA ACT AGT GGA TC
TAG AAG GCA CAG TCG AGG
CGC AAA TGG GCG GTA GGC GTG
CCG GGA GCT GCA TGT GTC AGA GG
GGG CTG GCA AGC CAC GTT TGG TG
GCT AGT TAT TGC TCA GCG G


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Cost of Service

ServiceMDACC Price/ReactionNon-MDACC Price/Reaction
Sanger sequencing-single reaction$5$10.25
Sanger sequencing-96 well plate$4$8.20


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Redo Policy

The facility will repeat a sample at no cost to the investigator if there is an instrument failure, or if the quality of sequence is compromised due to an error in the facility. If the investigator recommends a sample be repeated, the same DNA and primer will be used to repeat the sequencing reaction. If the reaction fails a second time, the investigator will be charged for the repeated reaction. If an incorrect primer has been requested for sequencing and as a result no sequence data is produced by the sequencing reaction, the cost will be borne by the investigator.

The facility will assist investigators in designing primers to difficult regions. Please contact Erika Thompson if you require this service

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Frequently Asked Questions

Why did my template not sequence?

The two most critical factors for the success of automated sequencing are DNA sample purity and concentration. The most common cause for reaction failure or poor quality sequencing results in our facility is DNA concentration. That's good to know because it is easy to fix! Right?

The DNA needs to come in at 100 ng/µl. We highly recommend that customers use either the QIAGEN plasmid DNA prep kits to purify their plasmid DNA samples or the Promega SV plasmid isolation kits.

Why should I resuspend my template in water?

The presence of salts or the EDTA in TE buffer will result in sequencing reaction failure.

EDTA is known to chelate magnesium and limit the activity of Taq DNA polymerase. We recommend that all templates be reconstituted in water. If you feel strongly about buffering your DNA you may store DNA in 10 mM Tris.

Does the host strain matter?

There are strains of E. coli that are better for plasmid production. Recommended strains are XL1-Blue, DH5(alpha), DH1, C600 SURE, NM294. Other strains such as JM 100 series, NM522, NM544, TB1, TG1, BL21, MC1061 do not produce good sequence quality DNA. These strains contain large amounts of carbohydrate that are released during cell lysis, and also contain an Endo A locus which results in the production of large amounts ofnuclease.

Does it matter which media I use to grow my cells?

Do not overgrow E. coli cells, especially in simple LB, as the cells decline rapidly after reaching stationary phase. Overgrowth can result in the early cell lysis, leading to the release of degraded chromosomal DNA and reduced plasmid quality. Standard LB with antibiotic selection is recommended for most protocols, with incubation of no longer than 16 hours to prevent cells from lysis.

My sequence stopped short. Why?

Please look at the sequence preceeding termination.

a) Is there a Poly A and/or T region? Frequently the polymerase will slip while trying to navigate these monomer repeats. This will result in the sequence becoming noisy on the other side of the repeat. The SMF has a poly T and a poly A primer to get through these regions.

b) Short tandem repeats, e.g. CTCTCTCTCTCTCTC, can cause the polymerase to terminate prematurely. The facility has several methods to try and navigate difficult regions of sequence. (Please talk to Erika if you have this kind of request.)

c) High GC content. This will frequently cause sequencing reactions to terminate due to hairpin loop formation within a sequence. The facility has a number of ways to navigate this kind of template depending on the sequence.

How Do I Get My Results?

Downloading Sequences

*Note: The text sequence provides you with unedited, raw sequence data. Please view the chromatograms to correct minor errors made by the base-calling software. The Sequencing and Microarray Facility provides a server for the rapid dispersal of data to the principal investigators. The results remain on the server for 30 days, after which files will automatically be deleted. Investigators should copy all data to their drives.

AppleShare


When using AppleTalk, the server will only allow 20 users to log on at a time. The server has been set up to allow 15 minutes per user log on to download files. If your workstation logs on automatically at start up you will be bumped off the system after 15 minutes.
(If you do not have a folder online, please contact the SMF and we will have a folder created for you.)

New Directions to Access Your Online Sequencing Results

PC users:
a) Click "Start"
b) Click "Run"
c) Type in “\\dcpavfs2a\seq”
d) Click "OK"
A dialog box will appear. Type in your "username" and "password".

Macintosh users:
a) Click on "Go" (located at top of screen)
b) Click on "Connect to Server"
c) Type in “smb://dcpavfs2a/seq”

Both PC and Mac users will use the same username and password that you use to access your MD Anderson account through Entourage and/or Outlook.

Viewing Chromatograms

FinchTV: This is a free application that can be downloaded in order to display and print chromatograms. Download from Geospiza.

Steps to download Finch TV:

1. Type the following into your web browser:
2. Click on Products
3. Click on FinchTV
4. Scroll to the bottom of the screen and click on “Download it today!”
5. Register your information
6. Follow the instructions on the screen

Step-by-Step Guidelines for Data Retrieval

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© 2014 The University of Texas MD Anderson Cancer Center