To ensure that individual primer walking projects are completed in a timely fashion we need to ask researchers to submit no more than two templates at a time for primer walking. Because of the volume of primer walking projects we are receiving, it is not possible to work on more than two projects from a single PI at the time.

Primer walking submission sheet

Sequencing by primer walking is an effective strategy for the efficient sequence determination of cloned DNA fragments which range in size from 0.5-4 kb. Initially sequence data is obtained using a standard primer which hybridizes to the vector sequence upstream of the insert DNA. Based on this initial data, a custom oligonucleotide is designed, synthesized, and used to prime a second sequencing reaction. The data obtained from the second reaction overlaps with the initial data and extends the sequence further downstream of the initial primer. By repeated cycles of custom oligonucleotide synthesis and DNA sequencing, the cloned insert is sequenced completely in one direction. Beginning with a second standard primer which primes in the other direction, this process is repeated to complete the sequence of the insert DNA on both strands.

Provide template at the appropriate concentration as outlined in the guidelines below. We then mix the initial primer and template for you and sequence it. Template should be provided with only sufficient volume for three times the sequencing reaction amounts. Our aim is for customers not to give us more sample than is necessary.

Excess template is not returned to the user but is discarded when the work is completed. Please send only sufficient material for the sequencing requested on each submission sheet.

Due to the volume of sequencing that we handle daily, if submissions are made with template provided at concentrations different from those above the sequencing may be significantly delayed. Thank you for following our guidelines carefully!

Please provide templates according to our guidelines as shown above i.e. 100-300ng/µl, 1µg per reaction. Please estimate how many reactions will be required - primers are designed at approx. 500 bp intervals. For example:

3kb insert: sequenced until data overlap: 6-7 reactions required: 6-7µg template at 100-300 ng/µl

Initial reactions may be sent as premixed reactions according to the premixed guidelines above and are charged at premixed rate, or the template may be sent at 100-300ng/µl, 1µg/reaction and the primer may be sent at 4µM, 3 µl/reaction for us to mix and charge at primer walking rate.

Sequencing by primer walking is a combined service of the oligonucleotide synthesis and DNA sequencing groups of the W.M. Keck Biotechnology Resource Laboratory:

• The user provides template DNA--double-stranded plasmid, single-stranded phagemid, or single-stranded M13 (10-20 µg plasmid DNA per kb of insert; Qiagen or CsCl preparations of plasmid DNA preferred).

• We perform sequencing reactions ($30 per template/primer combination), and design/synthesize custom oligonucleotide primers at a special reduced price (40 nmole scale; $0.83 per base + $3.74 setup).

• We return the sequencing data in a computer-readable format with a four color plot of fluorescent electrophoretic data, as well as custom primers. Usually, each researcher assembles the individual sequence files into a consensus sequence; sequence assembly and editing are available with an additional charge ($25 per kb of sequence).

The advantages of the combined service are:

• faster turnaround time (about 2-3 days between sequencing reactions)

• lower cost for oligonucleotide synthesis

• express service for oligonucleotide synthesis

Assuming a 1kb insert, custom primers 20 nucleotides long, and two sequencing steps in each direction (one per direction with a standard primer), then the total cost of the project would be:

(4 sequencing reactions X $30) + (2 primers X $20.40) which translates into a cost of $0.16 per finished base pair.

On average, the rate of data acquisition is about 1.2 kb per week.

If desired, sequencing may be performed in one direction only.