DNA Sequencing & Fragment Analysis

A mode cycle sequencing

In separate tubes, bring us your purified template (We do offer a template purification service.) and your custom primer, unless you wish to use one of our house primers (see list below). We will quantitate your template and prepare the sample for cycle sequencing and determine the sequence. Typical runs give high quality data out to >800 nts. The data is edited and emailed. A mode samples have the highest priority and are put at the head of the queue of samples.

B and C mode cycle sequencing

In a 1.5 mL eppendorf tube, add the proper amount of template and primer, qs with water (Do not use TE - the EDTA chelates Mg ion which is requirede by the polymerase). See DSEQ services for a more complete description of the different levels of service. Scroll down for a flowchart and for half and full plate preparation instructions.

This section contains instructions for getting a discount for half (36 to 71 samples) or full plates (72 to 96 samples). The 15% or 25% discount is applied if you follow these directions. Make sure sample names do not contain illegal characters (.\, /,:,*,?,",<,>,|.). If you have any questions regarding these directions please contact us before you prepare your plate. If you need plates for either mode we are happy to provide them.

C modes

1. Place samples in a non-skirted 0.2mL well volume pcr plate.
2. Add 300ng DNA and 3.2pmol primer in each well, q.s. to 10 µL with milliQ water
   
   • N.B., this is half the normal volume used with 1.5mL eppi tubes
   • for PCR or large templates this amount will vary,
     contact the core lab for more details.
3. Cover the plate and bring it to the lab (BSAC 6550)
4. We cycle the samples, purify and run the extension products on the ABI 3130xl sequencer and send you the data.

D modes

1. Use a half-skirted plate that says it is for use on the ABI 3100 & 3130 machine.
2. Cycle sequence and purify the dye-labeled extension products.
3. Speed-vac dry samples, cover with plastic wrap.
4. We run the extension products on the ABI 3130xl sequencer and send you the data.

For both methods

1. Samples must be arranged on the plate first TOP to BOTTOM,
   then LEFT to RIGHT.
   This means you must fill an entire column (A1...H1)
   then move to the next column (A2...H2), etc.
2. The default names reference the well positions, so it's easy to keep track of what's what if you loaded the plate as described.
   Note: you only need to put a single name for your plate in the template field on the standard workrequest very easy!
3. For "personalized" names (deprecated), you must fill out the spreadsheet that is available on the web.
   • In addition, please fill out the regular workrequest;
     it allows us to automate entry of the contact info.
   • Note: so for customized names you must send us two separate emails

You might want to check out these very useful notes on what can be a difficult, though time-saving, technique.

The key is to have a unique binding site for your sequencing primer. The ratio of primer to template needs to be correct too, so more template is not always better. Check that you have a unique product or use a nested, internal sequencing primer for the cycle sequencing reaction. To check this you need to run the products on a gel that has sufficiently good resolution. Call, 503-494-2442, or email Dr. Keller to discuss if you have questions or are having trouble.

This is the "do it yourself" mode: you do the cycle sequencing reactions using your own 'BigDye' reagents and purify the extension products. Then bring your dried down ready to load samples to us. We will add Hi-Di and run the sample on our machine. You do need approval from the core lab to use this service.

We offer a discount for high-throughput work: 15% off for half plates (36 to 71 samples) and 25% for full plates (72 to 96 samples). See the Plate Preperation protocol, contact the corelab for more information.

Special Chemistries are Available

We can sequence high GC content and other difficult plasmids and PCR products. We also have protocols for very large templates such as BACs. There is an additional charge due to the additional reagents that we use with these protocols.

Templiphi™ - Just give us a cloned (e.g. toothpick a colony) or a small amount of low yield culture, and we will amplify it using the rolling circle DNA polymerase from Phi29 to generate sufficient closed circular dsDNA to use for cycle sequencing.

Template amplification is free for A-mode samples, and only $5 for B-mode and C-mode samples.

A brief description from the Amersham manual

TempliPhi DNA amplification was developed by Amersham specifically to prepare templates for DNA sequencing. The TempliPhi method utilizes bacteriophage 29 DNA polymerase to exponentially amplify single- or double-stranded circular DNA templates by rolling circle amplification (RCA). This isothermal amplification method produces microgram quantities of DNA from picogram amounts of starting material in a few hours.

Amplification, in vitro, of very small amounts of template DNA eliminates the need for overnight cell culture and conventional plasmid or M13 DNA purification. The proofreading activity of 29 DNA polymerase ensures high fidelity DNA replication.

The starting material for amplification can be a small amount of bacterial cells containing a plasmid, an isolated plasmid, intact M13 phage, or any circular DNA sample. Bacterial colonies can be picked from agar plates and added directly to the TempliPhi reaction. Alternatively, microliter quantities of a saturated bacterial culture or a glycerol stock can serve as starting material. Depending on the source of starting material, amplification is completed in 4 to 18 hours at 30 ºC with no need for thermal cycling. The product of the TempliPhi reaction is high molecular weight, double-stranded concatemers of the circular template. Note that when starting with M13 clones, the TempliPhi product is double-stranded DNA and can be sequenced with forward and reverse primers. DNA amplified by the TempliPhi method can be used directly in cycle sequencing reactions without any purification.

The most convenient way to schedule DNA sequencing is the on-line work request. Email the Core Facility for more information.