Manager:
Pieter W. Faber, Ph.D.
Phone: (216) 444-7124
Email: faberp@ccf.org

Scientific Advisor:
Charis Eng, M.D., Ph.D., FACP

Sequencing:
Location: NE5-253
Phone: (216) 444-0974 (Xiaolan)

Illumina array services:
Location: NE5-253
Phone: (216) 444-7124

Genomics Core: DNA Sequencing

  1. Sample Preparation Guidelines.
  2. Sequencing Primers.
  3. Sequencing Plus Option
  4. Sample Submission / Data Retrieval Procedures.
  5. Pricing.
  6. Quality control / Re-sequencing.
  7. Core Contact Info.

Remember: High quality DNA equals successful sequencing.

The Genomics Core is equipped with a State-of-the-Art Applied Biosystems 3730xl DNA analyzer and employs experienced, well-trained personnel to handle sequencing projects. However, a major part of sequencing success in achieved at the bench where the template DNA is prepared and its concentration is measured: properly purified DNA preparations give good sequencing results. Optimal results are dependent on the absence of excessive salts, organic solvents and residual ethanol in the template DNA preparation as well as the correct amount of template DNA.

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1) Sample Preparation Guidlines

How to prepare high quality DNA sequencing template:

A) DNA isolation:

For plasmid preparations (either large-scale or mini-preps), numerous methods work IF you are sufficiently careful to avoid genomic DNA, excess RNA or contaminants. Many Core sequencing clients use Qiagen products with good success, but other manufacturer's products may work as well (contact the Core if you have specific questions). You can use Cesium Chloride preps, but make very sure there is no Cesium or EDTA present in the final sample. You may need to do an extra ethanol precipitation for this reason. Please contact the Core (faberp@ccf.org) if you plan to sequence large DNA templates (BACs, cosmids)

For PCR products, running the DNA fragments out on a gel and gel-eluting the desired band is preferred. Again, Qiagen products have been used with good success. Note that the PCR product must be VERY pure in order to obtain clean sequence. Non-specific bands may appear low-intensity, but could easily ruin the quality of the sequencing run. Also note also that all traces of the original PCR primers must be removed to avoid having them act as sequencing primers.

B) DNA Quantitation:

If you have enough template DNA, always determine its concentration by UV absorption. However, most of the time, you cannot reliably quantitate a mini-prep using a spectrophotometer! If OD260 reading are low (below 0.05) a better method to quantitate mini-prep DNAs is to run a small aliquot on a gel and estimate the amount of DNA by comparison with standards of similar size and known concentration. If you are unable to determine the concentration accurately, you should consider not proceeding until you have enough DNA to measure accurately.

C) Diluting the sequencing template to the correct Concentration:

After determining the template concentration, dilute the template to the correct final concentration (see below) using *distilled water* (please no TE or other EDTA-containing buffer), and avoid adding any divalent cations (i.e. Mg, Ca, Mn).

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2) Sequencing primers

 

GUIDELINES ON DESIGNING UNIQUE WORKING SEQUENCING PRIMERS:

Primer length: 20-25 nucleotides, Tm = 56-75 °C, G-C content: 40-60%, combination of two GC’s at 3’ end.

 

The Genomics Core currently accepts DNA sequencing templates and primers in three formats: 

  • Format A: DNA and primer in separate tubes
  • Format B: DNA and primer premixed (single sample)
  • Format C: DNA and primer premixed (full 96-well plate)

Format A) Core provided primers:

The Genomics Core currently provides the following primers for DNA Sequencing at no charge to you. To assure superior results and avoid delays, please confirm that the exact primer sequence is present in your vector.

  1. M13 Universal primer (-21):17-mer, 5'd(GTA-AAA-CGA-CGG-CCA-GT)3'
  2. M13 Forward (-20) primer: 18-mer, 5'd(TGT-AAA-ACG-ACG-GCC-AGT)3'
  3. M13 Reverse (-2) primer:17-mer, 5'd(CAG-GAA-ACA-GCT-ATG-AC)3'
  4. T3 Promoter primer 20-mer, 5'd(ATT-AAC-CCT-CAC-TAA-AGG-GA)3'
  5. T7 Universal-R1 primer 19-mer, 5'd(TAA-TAC-GAC-TCA-CTA-TAG-G)3'
  6. T7 terminator primer: 19-mer, 5'd(GCT-AGT-TAT-TGC-TCA-GCG-G)3'
  7. SP6-5 Promoter primer: 19-mer, 5'd(T-ATT-TAG-GTG-ACA-CTA-TAG)3'

These sequencing primers are available through the LRI storeroom in 50 µl aliquots at 5 pmol/µl which will facilitate submitting premixed template / primer solutions. See above for the sequences of these primers.

  • Lawson 211989:      T7 Universal-R1 primer
  • Lawson 211990:      T7 terminator primer
  • Lawson 211991:      SP6-5 Promoter primer
  • Lawson 211992:      T3 Promoter primer
  • Lawson 211993:      M13-universal
  • Lawson 211994:      M13-Foward (-20)
  • Lawson 211995:      M13-Reverse (-2)

Format A ) Client provided primers.

When using primers other than those listed above, provide at least 10µl of primer for every sample you are submitting at a concentration of 1 pMol / µl or 1 µM. Client provided sequencing primers need to be diluted in milliQ H2O to the required concentration. To dilute your primers to the required concentration, you may use the formula listed below:pmole primer = (primer in µg) x (1,000,000)(number of base pairs in the primer x 325). To verify that the above calculations are accurate, please measure the OD260 and use the following formula:C (pmol/µl) = A260 x 100 x dilution factor(1.54 nA + 0.75 nC + 1.17 nG + 0.92 nT)

Format B) Suggested amounts of primers in premixed samples.

When submitting premixed samples in individual tubes we need 11µl total with the following guideline amounts of DNA and primer:

DNA template Plasmid   PCR product
DNA amount 200 ng / 1 kb   20 ng / 100 bp
       
Primer amount 10 pmol   10 pmol
Alternative Primer Amount 100 ng   100 ng

When submitting premixed samples in 96-well plates we need 5 µl total with the following guideline amounts of DNA and primer:

Template Type Template Amount   Primer Amount Alternative Primer Amount
Plasmids (mini / maxi preps) 100 ng / 1 kb     5.0 pmol total 50-100 ng total
PCR  products / DNA fragments 10 ng / 100 bp   5.0 pmol total 50-100 ng total

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3) Sequencing Plus Option

Selecting this option is strongly recommended for larger sequencing templates as well as known GC-rich templates

Prior to November of 2006, the Genomics Core routinely used extra sequencing reagent in sequencing reactions involving templates larger than 9 kb as well as known GC-rich templates. This was done at no extra charge to customers and greatly improved the success-rate of the sequencing reactions involved.. Due to an increase in the amounts of such templates submitted to the Core for sequencing this service could no longer be continued free of charge. The use of extra sequencing reagent now is an option customers can select on the sample submission form for either all their samples or a subset of samples. The charge per sample for selecting this option will be $1.00.

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4) Sample Submission / Data Retrieval Procedures.

Use either the Sequence Submission form for individual samples or the Sequence Submission form for a 96-well plate.

As indicated above sequencing samples can be submitted to the Core in three different formats;

Format A: DNA and primer in separate tubes (link to submission form).

In this submission procedure clients provide 10 µl for each DNA template and each sequencing primer (if applicable) at the appropriate concentration to the Core in separate 1.5 ml reaction tubes. Core personnel will combine the DNA templates and primers and add the sequencing reaction mixture. The required concentrations of DNA template and primer are given below.

Template Type Template Concentration
Plasmids (mini / maxi preps) 200 ng / µl
PCR  products / DNA fragments
Small fragments (100-1000 bps) 20 ng / µl
Large fragments (over 1000 bps) 50 ng / µl
Large DNA templates       contact Core (faberp@ccf.org)  
Primer concentration      1 pmol/µl  

Every user must fill out a DNA Sequencing Request Form before submitting samples. The template and primer for each sequencing reaction should be listed clearly and have a unique name even if the sequencing templates are the same. The PI name is the owner of the listed activity number. To avoid loss or mix up of sample(s), it is advisable to tape the tubes securely on the form and enclosed them in an envelope. Samples are collected once a day between 9:00 a.m and 9.30 a.m. For your convenience there are a total of three sample drop-off areas on campus: at LRI they are in the small office corridor in the middle of the west NC1 hallway and in the 5th floor NB hallway, opposite the Chairman's office. For those who would like to drop off their samples in a refrigerated spot there is a Genomics Core refrigerator in the NN building: past conference room NC1-202 through the door marked "Area NN". For clients without CCF access there is a refrigerated drop-off point in the ND shipping and delivery area (small fridge on table).

Format B: DNA and primer premixed (single sample). (link to submission form)

In this submission procedure clients provide 11 µl of premixed DNA template and sequencing primer for each sequencing reaction at the appropriate amount to the Core in a 1.5 ml eppendorf tube (NO SMALL TUBES) (amounts are guidelines and clients should test what amounts work best for their specific templates and primers).

DNA template Plasmid   PCR product
DNA amount 200 ng / 1 kb   20 ng / 100 bp
       
Primer amount 10 pmol total   10 pmol total
Alternative Primer Amount 100 ng total   100 ng total

Format C: DNA and primer premixed (full 96-well plate). (link to submission form)

In this submission procedure clients provide 5 µl of premixed DNA template and sequencing primer for each sequencing reaction at the appropriate amount to the Core in a 96-well sequencing plate (amounts are guidelines and clients should test what amounts work best for their specific templates and primers).

Template Type Template Amount   Primer Amount Alternative Primer Amount
Plasmids (mini / maxi preps) 100 ng / 1 kb     5.0 pmol total 50-100 ng total
PCR  products / DNA fragments 10 ng / 100 bp   5.0 pmol total 50-100 ng total

Please contact the Core for a Sample Sheet when submitting sequences in the 96-well plate format. 96-well sequencing plates need to be delivered in person at NE5-253.

Data retrieval procedure

Electronic Sequencing data files (text and trace) are deposited on the LRI server Titan in the Folder labeled with the PI name on the day after the submission (an extremely busy sequencing schedule may delay this by a day).  We recommend the free software program Finch TV (Geospiza Inc) for the viewing of the electropherograms.

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5) Pricing

Sequencing: premixed template and primer $7.50 / sample
Sequencing: separate template and primer    $9.00 / sample
96-well plate sequencing  $480.00 / plate
Sequencing Plus option $1.00 / sample

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6)Quality control / Re-sequencing

A control pGEM-3Zf (+) standard DNA template is included with each set of sequencing reactions and run on the DNA analyzer to monitor the sequencing process. Failed reactions, which are due to Core associated issues such as gel anomalies are automatically repeated at no charge to the investigator. However, some sequencing reactions may fail due to template / primer associated issues such as: template-primer annealing problems, secondary structures, homopolymer repeats, GC compressions, G-stretches, or poor DNA preparations. In such cases the Core will work with the investigator to arrive at a working protocol that results in good sequencing results. Remember, our ultimate goal is to give you the highest quality of sequencing data that your sample allows.

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7) Core Contact Info.

DNA Sequencing Personnel:

Pieter Faber, PhD        Manager.

Xiao Lan Zhao             Sr. Technologist.

Lab phone: 216-444-0974

Office phone: 216-444-7124

 

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