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Molecular Biology Core Facility

Quillen College of Medicine

Sequencing

Using a Beckman CEQ 8000 automated DNA sequencer, the facility can get up to 800 bp of readable DNA sequence from plasmids, PCR products, phage or cosmid clones. Sequencing larger templates (BACs, YACs, etc.) is also possible. In combination with WellRed chemistry the facility can produce excellent DNA sequence data at a lower cost than using manual gels and radioactive labeling. Standard primers (T7, T3, SP6, M13F, M13R, etc.) are provided for free. Other primers are provided by user at 3 uM. Primer design services are available. Sequenced data are returned to the user by email within four business days, usually faster.

Clean template DNA

The DNA should be in dH 2O, although 10 mM TrisCl, pH 8 - 8.5 (Qiagen's EB) will also work.

DO NOT USE TE!!

Check the 260/280 ratio of your DNA. Samples with a ratio of 1.7 or less are not clean, and may not work! Please feel free to call us if you are unsure.

The sample MUST be clearly labeled with its name and concentration.

Please provide MORE than the minimal amount required for your sequencing reaction. We are happy to return any unused material to you, if you so desire.

Primer

If you require your own primer, please provide it, clearly labeled, at 3 pmole/l ( 3 uM) in dH 2O. We willneed 4 uL of 3 pmole/L primer per sequencing reaction. Please send a MINIMUM of 10 uL of primerin excess to the amount we should need for all of your sequencing reactions. Again, we are happy to return anyunused material to you, if you so desire.

Contact us if you need help in calculating the conversion ng/uL units into pmole/L units.

CLICK here for the Sequencing request form (PDF)

CLICK here for the Sequencing request form (DOCX)

 

  • Template size
  • Template concentration
  • Primer to use

An account number to bill.
An email address to send the results.

ANY SPECIAL INFORMATION that will aid us in sequencing your sample. For example, indicate if the is GC rich or will form secondary structures (shRNA vectors, recombination vectors).

Samples submitted to the Core Facility in STANDARD FORMAT*(see below)

By having all users submit their samples at a standard concentration we are able to automate the sequencing process thus significantly reducing preparation and reagent costs. It is recommended that sequences for publication be completely sequenced in both directions. Large template (YAC, BAC, etc.) sequencing is available at an increased cost.
Samples submitted to the Core Facility that are NOT in standard format are sequenced for $13/sample
Primers can be designed for $15/primer or $25/pair for regular (not real-time) PCR.
Plasmid mini-preps are $5/prep.
The costs are per sample (one direction).The sample will be re-run for free if there are any problems on our end with the sequencing run.

STANDARD FORMAT* FOR DNA QUANTITIES:

PROVIDE 10uL (or more)OF YOUR TEMPLATE. Amounts shown below are needed for sequencing one run, please allow enough for the number of runs you want for each sample (e.g. 2x for bidirectional) plus extra (just in case).

PLASMIDS: Provide at 100 ng/L. Any plasmid prep NOT provided at this concentration is NOT in standard format and we will charge you the higher sequencing price!


PCR PRODUCTS:
Refer to the chart below for estimation of PCR product needed per reaction; we generally use more PCR product than suggested on the table.

Size of Amplimer, bp
Total amount to submit per reaction
Sample concentration
100-200
10 - 20 ng
4 - 10 ng/uL
200 - 500
20 - 50 ng
10 - 20 ng/uL
500 - 750
50 - 75 ng
20 - 30 ng/uL
750 - 1000
75 - 100 ng
30 - 40 ng/ uL
1000 - 1500
100 - 150 ng
40 - 60 ng/uL
over 1500
150 - 200 ng
60 - 80 ng/uL

 

IMPORTANT NOTE!!!
The quality of your sequencing results will be directly proportional to the quality of your starting DNA. Criteria for cycle sequencing are more stringent than EITHER manual sequencing or standard PCR. The success of sequencing reactions depends critically on having high purity template at the correct concentration. The investigator is responsible for providing "sequenceable" template.

RECOMMENDED KITS FOR DNA ISOLATION/PURIFICATION
To aid the investigator in preparing high quality DNA, we routinely test different kits for their ability to REPRODUCIBLY generate high quality sample. We have sequenced literally THOUSANDS of samples and currently recommend the following kits:

ThermoFisher
SNAP Miniprep kits. PureLink Kits. Some of the longest sequencing reads we've obtained used these kits for the DNA isolation. ChargeSwitch Kits: One of the Director's personal favorites for ease and quality of the DNA isolation

QIAGEN
Qiagen kits routinely generate excellent quality DNA. Be careful not to have any leftover EtOH in your prep as this will "kill" the sequencing reaction.

YOUR SEQUENCE DATA
Your data willbe sent as a Compressed File orvia the ETSU dropbox.

VIEWING THE ELECTROPHEROGRAM
You can open with numerous viewing programs including, CHROMAS, VECTOR NTI, FinchTV and for MACs, EditView. These are free downloadable programs. Contact the Core Facility if you need assistance.

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