Sequencing Guidelines
Contents:
1.
Pricing
2.
Submitting Samples
3.
Primers for Sequencing
4.
Large Volume orders
5.
Samples Storage
6.
Results and Data Output
7.
Quality Control
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 : |
| $ 6.50 on campus investigators* |
| $ 12.00 off campus, in state |
| $ 12.50 out of state |
*Please contact the facility for volume pricing and other pricing considerations. |
We run positive controls with every sequencing and fragment run. 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. Back to top
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 .
If the website is down please contact the Facility at (406) 243-4229. An Excel format Order Form will be emailed to you, for your completion.
We can no longer accept handwritten orders.
a. Clearly label the TOP of each 1.5ml tube using the alphanumeric designation—3letter initials + a unique number. Example: "PMC34" Long labels 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. Please do not aliquot a single DNA sample into several tubes labeled differently, as this will only slow processing times.
b.On the Order Form, each sequence requested must be listed in the following format, exactly, templatename.primername. (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 note: M13F and M13R are just examples of a primer that may be used.)
c. Samples need to be in 1.5 ml microfuge tubes.
d. We need a volume of 10 to 15 µl at the concentrations listed in the table below for each sequence. (Example: two primers would be two sequences and require twice the volume (20-30ul); four primers would require four times the volume or about 40ul of template.)
e.Primers must be at 3.2pmol/ul. We will need at least 5ul of primer for each sequence it is used for.
DNA Type and special considerations : We need to know the type of DNA (plasmid, PCR product, low-copy plasmid, 12kb or larger), the concentration (see requirements below*), and any particulars such as high G-C percentages, AT rich, or repeats, which may be needed to determine if sequence is successful or if special reaction treatment is necessary.
Concentrations required for each template: (see 2d above for quantity/volume needed)
| Template |
Concentration |
| PCR product |
10ng/ul |
| Double-stranded DNA |
100ng/ul |
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 recommend 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.
Special Requests: (Please see section 4 below)
Our goal is to work with each client’s requests, if at all possible. Please contact us if you have a large order and would like to submit your samples in a 96 well plate, or with primer already added to template in the proper ratio, or if you wish to do the sequencing reaction yourself. We can discuss how to submit the order and any fee schedule changes that might apply.
For larger orders you can submit your sample via Excel. (We are working on getting this added to our online order form.)
Primers must be in 1.5ml tubes, 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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Please notify our lab ahead of time (2-3 weeks) if you are considering
undertaking a large project (>100) with the facility.
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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- The Murdock Lab will keep samples stores at -20C for 2-4weeks, depending on available space and throughput.
- Reruns need to be requested ASAP.
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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 AB Seqscanner (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, Data will be analyzed, and the chromatograms will be inspected, as will run logs for errors in the DNA sequencing process.
The Operator is not required to provide chromatogram editing or trouble shooting. 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 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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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.
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