DNA Sequencing
Guidelines

The quality of your sequence results will be directly related to the cleanness and correct concentration of your DNA and primer. With good quality DNA and primers, you should expect a 95% success rate. Low success rates are generally due to inadequate quantitation of templates and primers, as well as DNA that has been inadequately prepared. Several laboratories who use the facility have an outstanding success rate of 95-98%. Many have success rates at 85-90%.

We recommend Quiagen products for DNA preparation as being most reliable, or the alkali lysis/PEG precipitation (method in the protocol section). Please quality control the DNA by checking the O.D. and running the DNA out on an agarose gel next to a known DNA quantitation marker, BEFORE you submit the samples for sequencing. The Facility has plasmid DNA quantitation markers available for anyone who is experiencing a high rate of sequence failures. (Gibco BRL Cat #14420-012). These markers range from 15-500ng/6ul of uncut DNA and are ready to be loaded on a gel along with your samples. We also have PCR quantitation markers available for 50-1000 base pairs and 1000-2000 base pairs.

Check out the web site for information on DNA concentrations, template protocols, primer design/considerations, and sequence analysis.

TUTORIAL 4: for help with troubleshooting, includes :-

  1. Factors that affect the quality of sequencing.
  2. Types of difficult templates and solutions for sequencing them.
  3. Sequencing PCR products.

OFFICE HOURS: Margaret Robertson is usually available between 2.30pm and 3.30pm each day for consultation. If you cannot make it to the facility during "office hours" please call Margaret at 581-4736 to set up an appointment.

To Submit Samples for Sequencing

Customers should fill in a sequence request form, located in the top of the drop box outside of room 4A 437. Please fill in as much information as possible as this will help us decide the best possible sequencing chemistry and gel run for you. The insert size will help us decide what length gel to run. However, if you need only a few hundred bases, please tell us in the comments section. Generally a short run will generate up to 400 bases, a long run about 600-700 bases depending on the quality of DNA. Deposit the request slip in the bottom of the drop box. Make sure that you have an account number and a phone number clearly legible on the form. Deposit DNA and primers in the racks on the left hand side of the deli case, labelled : SAMPLES FOR SEQUENCING.

Sequencing can be done on many types of templates.The quality of your sequence will be directly related to the quality of your DNA template. Samples for sequencing should be assayed for quantity and quality BEFORE submission to the facility. Please provide the following templates and primers at the required concentration and volume. Submit the samples in a 1.5ml tube, labeled on the SIDE. Please keep sample names as simple as possible, less than 10 characters if possible, numerical and alphabetical only, dashes are ok to use. Avoid using ( ~ . * // ) in your sequence name. The volume requested is the minimum for one sequence reaction.

ALL DNA SHOULD BE IN WATER, AS THE PRESENCE OF HIGH CONCENTRATIONS OF EDTA WILL COMPROMISE THE CYCLE SEQUENCING REACTIONS!

ALL SAMPLES WILL BE DONE USING THE BIG DYE TERMINATOR CHEMISTRY (PE-APPLIED BIOSYSTEMS).

ALL DNA SAMPLES SHOULD BE PREMIXED WITH THE PRIMER IN A TOTAL OF 7UL.

Mix together the following concentrations of DNA and primer to give 7ul TOTAL:

300ng ss DNA with 3.2 pmoles primer.

600ng ds DNA with 3.2 pmoles primer.

30-60ng PCR product < 1Kb with 3.2 pmoles primer.

5-10ng/100bases of PCR product > 1Kb with 3.2 pmoles primer.

1ug of cosmid or P1 clone with 3.2 pmoles primer.

1-2ug BAC DNA with 10-20 pmoles of primer in a total volume of 12ul.

The facility will continue to provide -21 M13 FORWARD(UP), M13RP1(RP), T7, T3, SP6, T7 terminator and a polyT primer for cDNA clones. 4ul Aliquots of primers at 0.8 pmoles/ul will be kept in the freezer in room 4A437. Use 4ul in each reaction. A work area is available for customers to do any necessary mixing before submitting samples.

Please use 1.5ml tubes labelled on the side with the template name and on the top with the primer name.

PRIMER SEQUENCES FOR YOUR INFORMATION:

-21M13 (UP) 5'

TGT AAA ACG ACG GCC AGT 3'

M13RP1(RP)

CAG GAA ACA GCT ATG ACC

T7

TAA TAC GAC TCA CTA TAG G

T3

ATT AAC CCT CAC TAA AGG GA

SP6

ATT TAG GTG ACA CTA TAG

T7 TERMINATOR

GCT AGT TAT TGC TCA GCG G

POLY T (for sequencing through long polyT regions)

TTT TTT TTT TTT TTT TTT TTT TTN

COMPLETION OF SEQUENCES:

$10 will be charged for sequencing whether the reaction is successful or fails.Internal controls are on run on each gel .If a sequence fails due to problems within the core facility, we will, of course, repeat the reactions for no charge. Sequences can fail for many reasons. Dirty template, template concentration and primer problems are the most common reasons for failure. The facility staff can advise when you encounter problems, but check out our protocol section for advice on template preparation and primer considerations.

Generally, turnaround time is 2-3 WORKING days, but can be longer if the facility is very busy or has instrument problems.Samples will be sequenced in the order in which they were received with the following exceptions:

Medium size projects involving more than 50-100 samples at one submission, may be divided over several gels/days. Large projects involving hundreds of sequencing reactions will be done in a similar manner, but will generally take longer. If you have more than 50 samples contact the sequencing facility for a master plate layout and submit your samples in microtiter format. Primer walking projects can be done. At present there is no charge for this service, therefore turnaround time cannot be guaranteed. Consult with Margaret for an estimate for completion.

The facility staff will call the lab to let you know when sequence has been completed, but we cannot be responsible for messages not reaching you! Data will be distributed electronically. You must file an account request form (available from the sequencing facility) to obtain a user name and password to the HCI server. You will be able to log in and move your data to a local drive or server. We will no longer distribute data directly to your computer or on disk. This new system is more secure and much more convenient for everyone!

A hard copy chromatogram will be provided and can be picked up from the sequencing facility. These will be filed alphabetically by requestor's first name. You may check the chromatogram drop box any time to see if sequence has been completed, but please try not to interrupt facility staff more than is absolutely necessary to check on the status of your samples. We hope to have web access available soon to enable you to check the status of your project and to download your data.