Methods
Researchers at the Genetic Data Center select the best method for achieving the results required. Some of the methods we use on a regular basis are:
Isolation/Extraction
Many isolation techniques are used in the G.D.C. and each organism will be customized as to the best method for DNA or other macro-molecule isolation. Some examples of organisms encountered in the G.D.C.are: conifers, annual and perennial plants, birds, fish, insects, mammals.
DNA Isolation – Grinding plant tissues with liquid nitrogen
Markers
MJ thermal cycler
PCR based markers
Sequencing and Genotyping (SSRs, AFLP etc.)
Sequencing
The DNA Sequencers employed by the G.D.C. are the LI-COR® 4200 series automated sequencers. The capacity is 16 samples per channel for sequencing.
Sequencing Reaction
For the sequencing reaction, the DNA polymerase incorporates an infrared dye (IRD)-labeled primer into a set of chain-terminated fragments or the tailed sequencing method; the latter is the most commonly used method in the G.D.C. (see Steffans et al, Biotechniques 15:580-581, 1993). The IRD-labeled fragments separate according to size on a polyacrylamide gel. IRD labeled primers are sensitive to visible light. (Avoid extended exposure of IRD labeled primers to light by storing in the dark and wrapping primer stock tubes in foil for use on the benchtop.) Upon completion of the run, the sequence data can be presented as text (ASCII) or in curve format (standard chromatogram format, SCF) or as bands (tag image format, TIF).
Two different SequiTherm EXCELII Long-Read Sequencing Kits are available for use on LI-COR® Automated DNA Sequencers.
- “SequiTherm EXCEL™ II DNA Sequencing Kit-LC (for 66-cm gels)” This kit has termination mixes which permit read lengths up to 1000 to 1400+ bases.
- “SequiTherm EXCEL™ II DNA Sequencing Kit-LC (for 25-41-cm gels)” This kit provides darker signal intensities in the resolution range of 1-1000 bases. Users are those who routinely sequence templates of smaller size, such as PCR products or clones for verification.
| Sequencing | GDC |
| Sequencer | Li-Cor® 4000 and 4200 automated sequencers |
| Channel | IRD700 and IRD800 |
| PCR Machine | MJ thermal cyclers |
| Chemistry | SequiTherm EXCEL™ II DNA Sequencing |
| Sequencing Enzyme | SequiTherm EXCEL™ II DNA Polymerase |
| Primers | M13 tailed/end-labeled primers |
| Accuracy | greater or equal to 99% over 1000 bp |
| Template Material | ds DNA from plasmid (250-500 ng per reaction) |
| PCR product (125-250 ng per reaction) | |
| Template Preparation | Wizard® PCR Preps DNA Purification (for PCR products if necessary) |
| Primer Concentration | 1-2 pmol |
| Range | 75 to 1500 bp |
| Removal of excess terminations | Not required when using the tailed method |
| LiCor machine recovery | Size product >900 bp (66cm) |
| Size product <900 bp (66cm) | |
| Size product <650 bp (41cm) | |
| Size product <450 bp (25cm) | |
| LiCor data collection and scoring | e-Seq™ and AlignIR Alignment and Assembly Software™ |
All trademarks are property of their respective owners.
Product information, protocols and references can be obtained from the following web sites:
Sequencing from plasmids,
Sequencing directly from PCR products and Others:
Sequencing from dsDNA from plasmids
The quality and quantity of DNA templates is crucial in sequencing reactions. In terms of template added, please note that more is not better; excessive amounts of DNA in the sample can result in poor resolution, streaking, and lowered signal intensity or no readable signal. However, too little template will also result in poor resolution or premature termination of the sequence.
The SequiTherm EXCEL™ II kits are designed for plasmids, PCR products, and M13-based templates. Cycle Sequencing protocols are available at http://www.interscience.com/ and http://www.licor.com/. Larger templates like BACs, cosmids, and lambda phage can be sequenced with modifications in template concentration and number of cycles.
Sequencing PCR product directly
PCR products can easily be sequenced directly using the tailed primer method outlined above. The PCR product should be tested on an agarose gel and appear as a single band without any artifacts. Single band PCR products are the best templates for DNA sequencing. If most of the PCR primers are incorporated, the amplification product can be sequenced directly without purification. The PCR product can be purified by one of the following recommended methods:
- Wizard® PCR Preps DNA Purification
- QIAamp DNA Mini-kit
All trademarks are property of their respective owners.
Product information, protocols and references can be obtained from the following web sites:
Please contact us for other options such as:
Asymmetric DNA
Simultaneous bi-directional sequencing