DNA Sequencing (PCR and Plasmid)

Nanopore Sequencing

Utilizing Oxford Nanopore Technology sequencing platforms we have the ability to sequence entire plasmids and amplicons (>600 bp) without sequencing primers or PCR. Runs are performed Monday, Wednesday, and Friday. For technical questions and assistance, contact Graham Wiley.

DNA must be 30-50 ng/ul ≥ 10 ul and quantified using a fluorometric method (i.e. Qubit)
Files returned will include a .fasta sequence file and an .html summary file containing a complete annotation of each plasmid, along with read length and quality histograms
Sample Submission Cutoffs:
Samples for Monday runs must be submitted by 5 pm the previous Friday.
Samples for Wednesday runs must be submitted by 5 pm the previous Tuesday.
Samples for Friday runs must be submitted by 5 pm the previous Thursday.
Sample drop-off points are the refrigerators in rooms E324 and C102.
Request the service: (iLab)
Accessing your results: Select the “View My Requests” tab in iLab. The file is under your sample manifest.

*** OMRF will be retiring the 3730 Sequencer August 1***
The last run will be July 31. Samples MUST be delivered by July 30 in order to be processed!

Sanger DNA Sequencing

The OMRF Sanger DNA Sequencing facility provides low-cost Sanger method DNA sequencing to OMRF and OUHSC investigators, as well as investigators from other Oklahoma universities and schools. The Facility’s ABI 3730 capillary sequencer is capable of sequencing purified plasmid or PCR amplified DNA. Operating hours are Monday through Friday, 8:30 am to 4:30 pm. Beginning in September 2024 the sequencer will be run on Thursday. Most results will be ready the same day. Please deliver your samples by noon on Wednesday. The cost is $8.50 for OMRF, and $9.50 for Oklahoma educational institutions, out-of-state, and for-profit organizations. For best service, email Sheryl Christofferson.

Submissions

Submissions Worksheet

Concentrations and Volumes
Plasmid DNA: 100 ng/ul, 5 ul for each primer to be used
PCR DNA: 2 to 10 ng/ul depending on size, 5 ul per primer
Primers: between 3 and 5 uM, 5 ul per template
Sample drop-off points are the refrigerators in rooms E324 and C102.
Maximum read length per primer < 1kb

Primers

The Sequencing Facility provides the following primers:
T7, T7 terminator, T3, SP6, BGH Reverse, M13 Forward (-20), M13 Reverse (-27).

Primer sequences are:
M13 Forward GTA AAA CGA CGG CCA GTG
M13 Reverse GGA AAC AGC TAT GAC CAT G
T7 Promoter GTA ATA CGA CTC ACT ATA GGG
T3 Promoter CAA TTA ACC CTC ACT AAA GG
SP6 Promoter TAC GAT TTA GGT GAC ACT ATA G
BGH Reverse TAG AAG GCA CAG TCG AGG
T7 Terminator GCT AGT TAT TGC TCA GCG G

The Sequencing Facility welcomes custom primers that fit our guidelines.

have a T_m of 55^o-60^o C
length of approximately 18-24 bases
contain no mismatches
no secondary structure (particularly at the 3′ end)
no long strings (<4) of any single base, especially G or C

Analysis

DNA Viewer Links:
Finch TV (all platforms)
Chromas Lite (Windows)
4Peaks (Mac)

Contig Assembly Links:
Sequencher
DNA Baser

Accessing Results

OMRF Researchers Your sequencing data has been placed in a folder on an OMRF server. This folder is only for temporary storage of data.

Contact Sheryl at Sheryl-Christofferson@omrf.org, for information on accessing your data.

Mail samples to:
c/o Sheryl Christofferson
825 NE 13th Street
Mail Box 27
Oklahoma City, OK 73104

Sheryl Christofferson
Room E324
Sheryl-Christofferson@omrf.org