Murdock Lab

1. Pricing

The on campus rates are for those PIs who are directly associated with The University of Montana as faculty, staff, or student. All others are considered off campus users.

-- Sequencing :

$ 7.75 on campus investigators

$ 12.00 off campus, in state

$ 12.50 out of state

We run positive controls with every sequencing and fragment run. If we are unable to obtain quality sequence from our control, as well as from your DNA, you will not be assessed a charge or we will rerun your samples. However, if our control works we will expect payment regardless of whether your sequence worked or not. We will rerun any failed sequences deemed necessary by the investigator. If the sample gives the same results from an identical rerun, the account will be charged for both runs. If the sample gives good sequence from an identical rerun, there will be only one charge to the account. Please call if you have any questions.

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2. Submitting Samples

Sequencing

All orders must be submitted via this website. Following an initial account setup for each primary investigator's lab, all orders must be placed via this web page http://murdocklab.dbs.umt.edu/Order.aspx

For the present, you may be able to submit your orders via Excel spreadsheet format.    Please contact Patty at (406) 243-4229 for directions.

We can no longer accept handwritten orders.

Quantities and Labeling of Samples :

Templates brought in for cycle sequencing

2a. On the tube clearly label each tube alphanumerically (using your initials + a number) on the lid. (Example: "PMC34") Using three letter initials would be even better. Long names written on the tubes create confusion and are not acceptable. These must match the names on the order form.Do not include primer name with template name on template tube.

2b.On the Order Form, each sequence requested must be listed in the following format, exactly. Name of template.primer name. (See item 2a above.) Please note: there is a period (or, “dot”) separating the two names. So, someone named Patty McIntire would list her first sample with her first primer as “PM1.M13F” ,or another number of her choosing, such as “PM290.M13F”. If that sample is to be run with another primer then it would be listed again as “PM1.M13R”. Please call with questions.

2c. Samples need to be in 1.5 ml microfuge tubes.

2d. We need a volume of 10ul at the concentration listed in the table below FOR EACH SEQUENCE REQUESTED.  One sequence = DNA + primer.  So, if you have two primers please send 20ul of DNA.  If you have four primers to use with one template DNA, then send 40ul of template. 

2e.Primers must be at 3.2pmol/ul. We will need at least 5ul of primer for each sequence it is used for.

DNA Type : We need to know the type of DNA (plasmid, PCR product, low-copy plasmid), the concentration (see requirements below*), and any particulars such as high G-C percentages or repeats, which may be needed to determine if sequence is successful or if special treatment is necessary.

*Concentrations & Quantity required for each template:

Note: Generally plasmid sequencing requires more template, and a PCR product of 2kb or larger requires more product per reaction.

Sample Quality is paramount

All DNA, from 3kb plasmid vectors to 12 kb PCR fragments are acceptable. However, for efficient DNA sequencing, keep in mind that clean up of your sample is the single most important factor in receiving good sequence.

Sample Clean up: It cannot be stressed enough the importance of clean up. Any remaining ethanol or salts will prevent the sequencing reaction from proceeding. If you are using the Qiagen spin miniprep it is highly recommended that you adjust the protocol as follows: Following the addition of the Buffer PE and centrifuging for 30-60 seconds the protocol calls for you to discard the flowthrough and centrifuge for an additional 1 minute to remove residual wash buffer. We suggest discarding the tube as well as the flowthrough and using a new microfuge tube for the second spin. If you have questions, please call.

Many sequencing core facility websites (such as http://ecom2.mwgdna.com/improve_results_seq/improve_results.html and http://dnasc.byu.edu/indexResources.asp ) have recommendations for PCR fragment and plasmid purification. We have had particular success with templates from Qiagen and QiaQuick kits.

Samples already cycle sequenced, ready for clean up and electrophoresis:

Individual samples (up to 31) may be in PCR tubes, labeled numerically as clearly as possible.

Batches of 32 samples or more must be submitted in 96-well V bottom microplates, PCR plates, or .2ml strip tubes (8per strip).

Samples on 96-well plates will be loaded by column following the order: A1, B1 to H1, A2 so forth until H12. Follow the same format for partially filled plates and cross off any empty wells.

3. Primers for DNA sequencing

Primers must be diluted in water and provided at a concentration of 3.2pmol/ul.  Otherwise, denote a universal* primer to be used.

*Universal Primers supplied by the Facility:

M13F(-40) 5'GTA AAA CGA CGG CCA GT3'

M13R(-24) 5'AAC AGC TAT GAC CAT G3'

M13R(-48) 5'AGC GGA TAA CAA TTT CAC ACA GGA3'

T7 5'TAA TAC GAC TCA CTA TAG GG3'

SP6 5'ATT TAG GTG ACA CTA TAG3'

T3 5'ATT AAC CCT CAC TAA AGG GA3'

Please send at least enough template and primer for 4 reactions and already diluted to the concentrations listed above.

Primer Design: The following recommendations are provided to help optimize primer selection:

• Primers should be at least 18 bases long to ensure good hybridization.
• Avoid runs of an identical nucleotide, especially runs of four or more Gs.
• Keep the G-C content in the range of 30-80%, preferably 50-55%.
• For cycle sequencing, primers with Tm45 C produce better results than primers with lower Tm.
• For primers with a G-C content less than 50%, it may be necessary to extend the primer sequence beyond 18 bases to keep the Tm >45C.
• Use of primers longer than 18 bases also minimizes the chance of having a secondary hybridization site on the target DNA.
• Avoid primers that can hybridize to form dimers.
• Avoid palindromes because they can form secondary structures.

 

Primers manufactured by reputable, commercial, oligonucleotide synthesis vendors are acceptable. Primers manufactured "in house" on personal oligo synthesizing equipment are potentially suspect.

Fluorescent automated DNA sequencing is very sensitive to primer/oligo synthesis by-products including, but not limited to, salt contamination. Most commercial vendors supply primers in a highly purified state.

The Facility also recommends computer design of oligo sequences. Primers designed by eye often do not take into account competing priming sites, G+C content, TM, hairpin, self-duplex, or self-3'-dimer effects. We currently recommend using http://www.basic.northwestern.edu/biotools/oligocalc.html or similar web page to aid in design.

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4. Large volume sequencing

Please notify our lab ahead of time (2-3 weeks) if you are considering
undertaking a large project (>100) with the facility.

Large Orders of >50 samples: You may submit samples in a 96 well plate. You may also submit the order in Excel format--See example below. Submit your order via email attachment to pattymc@mso.umt.edu with the subject line reading “Murdock Lab – Order Placed”

Primer walking projects. The user is expected to analyze output chromatograms, design primers and supply those primers to the Facility for further sequencing.

Samples will be processed according to submission order, but very large submissions will be gradually processed at the discretion of the facility to accommodate the needs of smaller users

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5. Sample Storage

• The Murdock Lab will keep samples stores at -20C for 2-4weeks, depending on available space and throughput.
• Reruns need to be requested ASAP.
• Primers will be kept stored on a space available basis.

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6. Results and Data Output

RESULTS : You will be notified via email when your sequencing is complete and the sample files have been uploaded to the ftp server. You can download your Sequencing files at that time and view them using the EditView software (Mac users) or the Chromas software (PC users).
Your samples will be kept in freezer storage for 2-4 weeks following the run and then discarded unless we hear from you before then.

Data will be supplied in non-proofed (raw data) Applied Biosystems sample files. Upon completion of a DNA sequencing run, chromatograms will be inspected, as will run logs for errors in the DNA sequencing process.

The Operator is not required to provide chromatogram editing, trouble shooting or analysis. Services rendered in this regard are at the discretion of the Operator. Information about programs for viewing and editing chromatograms will be provided upon request. It is up to the client to determine the end point for reliable sequence by reviewing the chromatograph and/or raw data supplied.

FTP Server

All sequencing results are uploaded to the ftp server: ftp://dbssrv5.dbs.umt.edu, as soon as they are complete. You will be issued a user name and password to access your data. All data is uploaded in PC format. To convert to MAC format, download the conversion software provided free from Applied Biosystems.

We will keep sample files on the ftp server for download available for 1 month. After 1 month data will be deleted. Users are responsible for backing up their own sequence data.

Troubleshooting assistance

Many core facilities and commercial sequencing centers have websites which offer assistance for troubleshooting problem sequences. Two that we have found most useful for our clients are: http://ecom2.mwgdna.com/improve_results_seq/improve_results.html and http://dnasc.byu,edu/indexResources.asp .

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7. Facility Quality Control

Quality Control during DNA sequencing activities is an ongoing dynamic process. The Operator will make every effort to maintain and operate the facility at a high level of quality and accuracy. We welcome courteous suggestions and will attempt to accommodate requests. We appreciate your involvement and await your sequencing requests.

 

8. New Methods Policy

The Murdock Facility supports novel uses of DNA analysis, but not the development of novel protocols.  Users who wish to use the Facility for novel methods need to provide a functioning protocol (one that they or other UM researchers have used in the past to produce usable results), and pay for additional costs of optimization, as well as actual running expenses.  Optimization of novel uses should not compromise the operation of the Core facility functions to existing users.

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