SEQUENCING CHEMISTRY - FREQUENTLY ASKED QUESTIONS
Q: What does the DYEnamic™ ET terminator cycle sequencing kit offer me that I cannot achieve with BigDye™ terminator kit?
A: The DYEnamic ET terminator cycle sequencing kit offers:
- Shorter cycling time (1 min vs. 4 min)
- Excellent performance on difficult templates (GC-rich, tandem repeat, etc.)
- Salt tolerance (100 mM compared to less than 50 mM)
- Easy post sequencing clean-up (fewer dye blobs)
Additionally, Amersham Biosciences has considerable expertise in the field of separation media. As an alternative to ethanol precipitation, sequencing samples can be purified using gel filtration columns. For example, the AutoSeq™ 96 Plate uses a combination of chromatographic separation media (Sephadex™ G-50), PVDF membrane, and centrifugation in a 96-well format to remove excess salts and unincorporated fluorescent ddNTPs. The sequencing chemistry and the post sequencing clean-up reagents are offered in a cost-effective bundling price.
Q: How easy is it for me to set up the ABI Prism™ 3700 to use DYEnamic ET terminators?
A: The task of preparing the ABI 3700 to use DYEnamic ET terminator chemistry is relatively simple.
- First, perform a spectral calibration using the DYEnamic ET matrix standard for the ABI 3700 (US84001). This will create a new spectral calibration matrix for DYEnamic ET dyes. A simple and easy to follow procedure is included with the matrix standard.
- Next, download the DYEnamic ET terminator mobility files onto your computer. The mobility files are included on a CD (US84003) or can be downloaded here. Simple instructions ("readme" file) for installing the mobility files are included.
- The run module does not need to be changed. However, select Dye Set F and the correct ET terminator mobility file when setting up the ABI 3700.
- Finally, prepare the sequencing reactions with DYEnamic ET terminators following the protocol booklet provided.
Note: Use of the DYEnamic ET terminator cycle sequencing kit will require that new cycling conditions are programmed into the thermal cycler. Recommendations for cycling conditions are included in the sequencing kit protocol.
Q: Can the DYEnamic ET Terminator Cycle Sequencing Kit be used with ABI 3100?
A: Yes. The steps for preparing the ABI 3100 to use DYEnamic ET Terminator sequencing reagents are very similar to those required for the ABI 3700.
- First, order the DYEnamic ET Terminator Matrix Standard for the ABI 3700 (US84001) and perform a spectral calibration. Please refer to the "readme" file of ABI 3100 mobility file (Link to ABI 3100 mobility file readme section) for detail protocol.
- Next, download the DYEnamic ET Terminator mobility files for ABI 3100 or obtain it through contacting your local Amersham Biosciences Technical Service group.
- The run module does not need to be changed. However, select Dye Set F and the correct ET terminator mobility file when setting up the ABI 3100.
- Prepare the sequencing reactions with DYEnamic ET Terminators in following the protocol booklet provided.
Q: I'm a new customer who has never used DYEnamic ET Terminator Kit on the ABI Prism instrument before. What are the products that I'll need to order for the first time set up before testing the kit?
A: If you are using ABI 310, ABI 373 or ABI 377 instrument, you can order the ET Terminator Install Kit for ABI 310/373/377 with the product code 25600377. It includes the required mobility file, DYEnamic ET Terminator Matrix Standards, loading solution and one 100-reaction pack size DYEnamic ET Terminator Kit for these instruments.
If you are using ABI 3100 and/or ABI 3700 instrument, you can order the ET Terminator Install Kit for ABI3100/3700 with the product code 25603700. It includes the required mobility file, DYEnamic ET Terminator Matrix Standards, loading solution and one 1000-reaction pack size DYEnamic ET Terminator Kit.
Q: Is it necessary for a service technician to make a spectral calibration matrix?
A: No. The procedure for creating a spectral calibration matrix with the DYEnamic ET Terminator Matrix Standard is straightforward and easy to follow. Most ABI 3700 and ABI 3100 users have successfully performed this task without difficulty. If you need assistance please contact your local Amersham Biosciences representative.
Q: Can both DYEnamic ET Terminator and BigDye terminator sequencing products be run together on the same slab gel?
A: Yes, you can simultaneously use DYEnamic ET Terminators and the BigDye terminators on the ABI 377 and ABI 373 systems; they can be run together under Filter Set E.
Q: Can both DYEnamic ET Terminator and BigDye terminator sequencing products be run together on the same capillary instrument (ABI 3700 or ABI 3100)?
A: Yes, just be sure to select the correct dye set and mobility file for each sample.
Q: How will the use of DYEnamic ET Terminators affect my warranty with Applied Biosystems?
A: Please read your warranty terms carefully. In the past customers have used DYEnamic ET Terminator sequencing reagents on the ABI 373, ABI 377 and ABI 310 systems without invalidating warranties.
Q: Can the DYEnamic ET Terminator premix be diluted?
A: Yes. For the DYEnamic ET Terminator sequencing products, the most consistent results are obtained using 8 µl of sequencing reagent premix in a reaction volume of 20 µl, as described in the sequencing kit protocol book. These are the recommended conditions for routine use and should be the starting point for any optimization that the customer chooses to perform.
Some customers may choose to sacrifice some consistency in data to achieve greater economy. If this decision is made, DYEnamic ET Terminator Dilution Buffer (product code US84002) will provide the appropriate buffer conditions, salts, metal cofactors, and stabilizing agents necessary for optimal enzyme activity. In any case, the level of dilution that produces acceptable data must be determined empirically by the customer.
Please note that Amersham Biosciences makes every effort to develop products that are consistent in performance over a broad range of conditions. We do NOT recommend or support dilution of our products, and we make no guarantee concerning the extent to which a product can be diluted. We provide DYEnamic ET Terminator Dilution Buffer simply as a courtesy for those customers who wish to economize, with the understanding that the customer must accept the risk of reduced performance with dilution.
Q: Why is the price of the DYEnamic ET Terminator Dilution Buffer so high?
A: The DYEnamic ET Terminator Dilution Buffer is priced competitively.
Q: What is the composition of the DYEnamic ET Terminator Dilution Buffer?
A: The formulation of the DYEnamic ET Terminator Dilution Buffer is proprietary.
Q: What additional protocols do you have for large template DNAs such as BACs?
A: Recommendations for sequencing larger templates are made in the protocol booklet on page 24.
Q: How close to the primer can sequence be determined with DYEnamic ET Terminators?
A: The DYEnamic ET Terminator Cycle Sequencing Kit and the protocols and mobility files developed for it are optimized to provide the highest quality sequence. When used accordingly, base calling can start within 20-30 base pairs from the 3|-end of primer with most sequencing systems.
Q: Is the loading solution used on the slab gel systems the same as for the capillary systems?
A: No.
- For slab gel systems, we provide Formamide Loading Dye (US79448), a colored loading solution facilitating easier gel loading. This solution contains a higher salt content that will interfere with electrokinetic injection into capillaries.
- For the ABI 3700, ABI 3100 and ABI 310 systems, we recommend you use the MegaBACE™ Loading Solution (US79916).
Q: Why is the red loading solution in the kit if I cannot use it on the ABI 3700 or ABI 3100?
A: Since we did not change our product code, we kept the Formamide Loading Dye (US79448) in the 100 reaction (US81050) and 1,000 reaction (US81060) kits for the convenience of slab gel users.
For the ABI 310, ABI 3100 and ABI 3700 systems, customers may order the MegaBACE Loading Solution (US79916) separately.
Q: If I use the Formamide Loading Dye included in the kit with the capillary instrument will you replace the kit?
A: No. A special warning card is included in the kit and the protocol clearly explains the appropriate loading solution required for each sequencing system.
Q: What is the recommended loading solution for ET terminator 384-well plates?
A: We recommend MegaBACE Loading Solution (US79916) for all capillary electrophoresis instruments whether using a 96-well plate or a 384-well plate.
Note: After approximately 15 hours some degradation of samples stored in formamide will be observed.
Q: With respect to spectral calibration, what is the acceptable range of ET terminator condition numbers for the ABI 3700 and ABI 3100?
A: The condition number is a measure of the fluorescent dye spectral overlap. The SeqStd(AnyDyeSet).par we use to calibrate the instrument does not take into account condition numbers when assigning a spectral calibration. The condition numbers for ET terminators will be higher than the default parameter files. You should expect condition numbers of approximately 7.3 ± 0.5.
Q: What is the minimum Q value for ET terminators for the ABI 3700 and ABI 3100?
A: The Q value is a measure of how well the final matrix fits the data from which it was computed. A Q value of 1.0 is a perfect fit. Any spectral calibration having
a Q value of less than 0.92 will automatically fail the SeqStd(AnyDyeSet).par calibration. Typically, we see Q values of 0.95 and above with ET terminators.
Q: What recommendations do you make for sequencing with 384-well plates?
A: For optimal results, it is important to maintain the appropriate concentration of buffer components in the sequencing reaction. To achieve this, we recommend that 40% of the final reaction mixture be premix. If the total volume of the sequencing reaction mixture is less than 20 ml then all of the components of the reaction should be scaled appropriately. For example, if 5 ml reactions are assembled in a 384-well microplate, then 2 ml of ET terminator premix would be required in each sequencing reaction to achieve a 40% concentration of premix in the final reaction volume. Recommendations for precipitation in 384-well plates can be found on page 7 and in Appendix 8 (page 24-25) of the DYEnamic ET Terminator Sequencing Kit protocol. | 
