High-Throughput DNA Sequencing and Genotyping Core Facility

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DNA Sequencing Sample Recommendations November 10, 2010

For customers located at the UNMC campus, we strongly recommend the internet-based DNAlims system to submit the sample information as well as download the results which can be found at DNAlims.

For customers that are not located at the UNMC campus, please print off the PDF DNA Sequencing Order Form and complete prior to sample submission. To print the form you will need Adobe Acrobat reader which is available as a free download from Adobe.

The facility routinely sequences DNA fragments contained in plasmids, PCR products etc. Please read this information for important sequencing tips prior to bringing your samples to the facility. This will help to ensure the quality of your sequencing results.

DNA Template and Primer Quantities Required for Sequencing:DNA Template and Primer Quantities Required for Sequencing: The DNA sample should be provided to the lab premixed with the appropriate DNA sequencing primer in a PCR strip tube in a total volume of 20 μl. For standard DNA sequencing primers to include M13F-21, M13R-27, M13F-40, M13R-48, T7, T3, SP6, BGHR, T7terminator, the UNMC DNA Sequencing Core will provide aliquots of the primer to individual labs. For other DNA sequencing primers, the individual labs will continue to be responsible for them. We will provide assistance regarding primer dilutions as requested.

Listed below are the details regarding how to provide your samples in a PCR strip tube containing your DNA and primer.

See details below.

The DNA template plus primer must equal 20 μls of total volume. In all cases, we require 12.8 pmols/20μl. For example, add 4 μl of 3.2 pmols/μl of primer to make a final concentration of 0.64 pmols/μl of primer.

Template Primer
PCR Products
100-200 bp

200-500 bp

500-1000 bp

1000-2000 bp

>2000 bp

4-12 ng/20μl

12-40 ng/20μl

20-80 ng/20μl

40-160 ng/20μl

80-200 ng/20μl

Single Stranded DNA 100-200ng/20μl
Double Stranded (plasmid) 600-1200ng/20μl
Cosmid, BAC 2-4μ/20μl
Bacterial Genomic DNA 8-12μg/20μl

Cosmid, BAC and bacterial genomic DNA are problematic to sequence. If you need to have sequencing done on these types of templates, please contact the core director, Dr. Jim Eudy, beforehand.

For more details regarding mixing your DNA template and primer samples, please contact the core facility as necessary.

Sequencing Primers: The core facility provides commonly used primers at no charge.

Primer name Sequence
M13 Forward (-20) 5.-GTA AAA CGA CGG CCA GT
M13 Forward (-41) 5.-CGC CAG GGT TTT CCC AGT CAC GAC
M13 Reverse (-27) 5.-CAG GAA ACA GCT ATG AC
M13 Reverse (-48) 5.-AGC GGA TAA CAA TTT CAC ACA GG
T3 5.-AAT TAA CCC TCA CTA AAG GG
T7 promoter 5.-TAA TAC GAC TCA CTA TAG GG
T7 terminator 5.-GCT AGT TAT TGC TCA GCG G
SP6 Promoter 5.-TAC GAT TTA GGT GAC ACT ATA G
BGH-R 5.-TAG AAG GCA CAG TCG AGG

Submission of the Samples to the DNA Sequencing Lab:

  1. Label sample tubes clearly with the Request# for each sample. The Request# will be assigned by our dnalims system when you enter your order.

  2. Prepare your samples by adding the appropriate amount of template/primers to water (i.e., not TE or Tris buffers).

  3. The DNA concentration is a very important parameter with respect to the quality of your sequence data. Please make your measurements as accurately as possible, especially when submitting PCR fragments. It is common to overload the template amount resulting in a failed reaction. When working with plasmid DNA, please do not provide samples < 40 ng / μl.

  4. If for some reason you need to submit your DNA template samples and primers separately, please use our preferred primer concentration: We prefer 3.2 μM (3.2 pmol/μl) in H2O.

  5. Please submit the samples before 2:00pm in order to get the results next day.

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