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 µ M.  Primer design services are available. Sequenced data are returned to the user by email within four business days, usually faster.

FEES:

Samples submitted to the Core Facility in STANDARD FORMAT (described below) are sequenced for $8/sample.

  • 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.


Submission Guidelines
You Provide:

1. Clean  template DNA in amounts shown below.


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.



2.  A completed sequencing request form. Please click here for the form.  Include on it:

-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).

3.  If you require your own primer, please provide it, clearly labeled, at 3 pmole/µl  ( 3 uM) in dH 2O. We will need4 µL of 3 pmole/µL primer per sequencing reaction.  Please send a MINIMUM of 10 µL of primer  in excess to the amount we should need for all of your sequencing reactions.  Again, we are happy to return any unused material to you, if you so desire.

Contact us if you need help in calculating the conversion ng/uL units intopmole/µL  units. 


STANDARD FORMAT FOR DNA QUANTITIES

WE WILL USE 2.5 µ L OF YOUR TEMPLATE IN EACH REACTION; therefore, for optimal results please follow the submission guidelines.  A mounts 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/µL.   Any plasmid prep NOT provided at this concentration is NOT in standard format and we will charge you the higher sequencing price!  Contact the Core Facility if your plasmid is larger than 8 KB.

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:

INVITROGEN CORP.
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 CORP.
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 will be emailed to you through the ETSU Dropbox as an .scf file.

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.