The purity and concentration of the DNA template are the two most critical factors for obtaining good quality sequencing data. The increased sophistication of modern sequencing instruments such as our ABI3730xl allows for improved DNA sequencing but it also increases sensitivity to contaminants in DNA samples. A properly prepared and purified DNA template will result in maximal read lengths, higher sample score values and fewer errors, thus ultimately a quicker turn-around-time.
Plasmid Prep Cleanup Considerations:
Plasmid templates can be affected by a variety of contaminants such as cellular components (e.g. RNA, proteins, and polysaccharides), salts, organics or ethanol from commercially-available kits or precipitation method. Below are suggestions for producing a high quality preparation of plasmid DNA for a sequencing reaction. When eluting your samples, we recommend eluting in molecular-grade water.
RNA: The presence of RNA in a sample can interfere with proper quantification of DNA when using a UV spectrophotometer. Similar to DNA, RNA absorbs 260nm light and can result in overestimation of DNA quantity leading to poor sequencing reads.
SALTS: The presence of salts can decrease the processivity of the DNA polymerase used in the sequencing reaction. Care should be taken when precipitating DNA with alcohol to ensure that supernatants are completely removed and sufficient washing with 70% ethanol is performed.
EDTA: EDTA is a chelating agent and can sequester the magnesium required by the polymerase for the sequencing reaction thereby inhibiting its activity. To ensure EDTA is not interfering with the reaction is to provide your samples in sterile nuclease free water.
ETHANOL: The presence of low amounts of ethanol (10%) often causes the sequencing reaction to fail. Take care to avoid transferring ethanol from buffers used in isolation kits to the final elution and to completely drying precipitated DNA samples.
PHENOL: The presence of phenol following DNA extraction will denature the DNA polymerase used for the sequencing reaction. If using a phenol-chloroform extraction protocol take extra care to collect only the aqueous phase.
PCR Products Cleanup Considerations:
Sequencing of PCR products can be affected by contaminants such as unused primers and unincorporated dNTPs. Unused PCR primers can result in multiple peaks and a ‘dirty’ sequence read while excessive dNTPs can lead to an unbalanced dNTP/ddNTP ratio, resulting in potentially poor peak height. Below are some suggestions for generating a high quality PCR product for use in a sequencing reaction.
- Ethanol Precipitation of DNA by by Sambrook J., Russell D.W., Molecular cloning - a laboratory manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001
- Qiaquick PCR Purification kit. Website: http://www.qiagen.com/
- ExoSAP-IT® For PCR Product Clean-Up: an exonuclease/shrimp alkaline phosphatase treatment Website. http://www.affymetrix.com/estore/index.jsp (Type PCR Product Purification in Search)
Concentration of the DNA template is also very important for the sequencing reaction. Both excessive and insufficient quantity of DNA can lead to reaction failures or mixed results. Two of most commonly used methods for quantifying DNA are spectrophotometry and gel electrophoresis.
Quantification By Spectrophotometer
When using the spectrophotometer, take OD readings at both 260 nm and 280 nm. Absorbance of DNA at 260 nm should be between 0.2 and 1.0. RNA contamination can be determined by calculating A260/A280 ratio, where optimal ratio should be between 1.7-1.9. A wavelength scan from 220 nm to 330 nm can sometimes be indicative of other contaminants (e.g. phenol). To calculate DNA concentration (ng/µL), multiply Absorbance Value by Conversion Factor (50ng/µL) by Dilution Factor. Note: In our lab, we use the NanoPhotometer® Pearl P330 for plasmid DNA and PCR product quantification. However, for PCR product quantification, we first purify using QiaQuick Purification to avoid false and inaccurate readings to avoid over-estimation of concentration.
Quantification By Gel Electrophoresis
Quantifying by agarose gel electrophoresis provides an approximate quantification of double-stranded DNA. Run a DNA ladder beside your DNA sample to determine the concentration. For a rough estimate, visually compare your template band to the ladder band with a similar intensity and use the information provided by the ladder supplier to calculate your concentration. A more accurate estimation of DNA quantity can be obtained by calculating with band intensity values obtained using computer software.
Note: Primer quantification is equally important.
How much DNA template and Primers to submit:
- We required 5-10uL of DNA template and custom primer. More is always better!
- Refer to the table below for the concentrations required:
Type of DNA Length Concentration PCR products "<"499 bp 50-100ng/µL PCR products 500-1000 bp 100-150ng/µL PCR products >1 kb 60-180ng/µL PCR Plasmids All 60-200ng/µL Primers - 30-40ng/µl
Where to submit
700 Bay Street, Suite 1100
Toronto, Ontario - M5G 1Z6
Please follow these instructions when shipping DNA samples to ACGT corp.
- ACGT accepts samples for sequencing from 9am to 5pm Monday through Friday (for next day turn-around).
- Ship at room temperature. No need for dry or wet ice necessary.
- Remember: Label tubes clearly and cap tightly to avoid contamination or errors.
- Complete DNA Sequencing Request Form. If you forget, no worries, we can help at the office. Forms also available on-line.
Sample Pick up
To schedule a sample pick-up, please fill out the form at acgtcorp.com/schedule-pick-up. Your request must be submitted by 11:30am for same-day pick-up. Please have the samples and completed DNA Sequencing Order form ready for pick-up. Sample pick-up is offered free of charge from the following locations:BEST Institute – 112 College Street
CCBR – 160 College Street
Faculty of Pharmacy – 144 College Street
Fitzgerald Bldg – 150 College Street
HSC – 555 University Avenue
MSH – SLRI at 600 University Avenue & TCP at 25 Orde/60 Murray Street
MSB – 1 King’s College Circle
PMH / OCI – 620 University Avenue
Tanz Neuroscience Bldg – 6 Queens Park Crescent
TMDT & MaRS South Tower – 101 College Street