DSEQ Sample Preparation
Cadillac, Full Service (A-MODE)
We now offer a new sequencing chemistry for difficult templates including G/C rich templates, homopolymers, repetitive sequences and secondary structures.
In separate tubes, bring us your purified template (We do offer a template purification service.) and your custom primer, unless you wish to use one of our house primers (see list below). We will quantitate your template and prepare the sample for cycle sequencing and determine the sequence. Typical runs give high quality data out to >600 nts, and "good" templates will give reads beyond 800 nts. The data is edited and emailed. A mode samples have the highest priority and are put at the head of the queue of samples.
Hybrid Service (B-mode)
For each B mode sample, check the Template Quantity table and bring this amount of template in a 1.5 mL eppendorf tube along with sufficient quantity of milliQ water to bring the volume of 16 µL.
We will add either 4µL of the "House Primers" you wish to use, or your custom primer at 3.2 pmole/µL (µM). So the final volume is 20 µL.
We will use 10µL of the final 20µL (template+primer+water sample), add our BigDye/Better Buffer master mix and cycle as appropriate for your sample. We purify the fluorescent extension products and run them on our AB capillary 3730xl.
We will send you the chromatograph and text file, and will edit them if requested. Be sure to edit your sequence data: especially the low signal regions at the start and end of the runs.
For experienced sequencers: just give us 20 µL of a 2X sample solution (dsDNA template + primer + water) in a 1.5 mL 'eppendorf' tube. We will cycle, purify and sequence. You edit and troubleshoot your own sequence data, ... so wear your helmet!
If you have lots of samples, but don't want to cycle them yourself (D-mode), you can submit them in a plate. But in this case, we just need 10 µL in each well containing 300 ng template and 3.2 pmole primer. Contact the lab for help, or download the D-mode 96-well plate protocol and follow it through step 2.
Further data processing and analysis is available on a fee-for-service basis. Also, any samples requiring extra reagents will be considered B-mode.
You will receive sequence data, ready for editing.You should edit the base calling for each run before assembly or further analysis. Especially note that the ends of sequences usually have low signal, hence low quality, and should be truncated.
Template & Primer Amounts
|Size of plasmid (kb)||Template Amount (ng)||Primer amount (pmole)||Total Volume*|
|L < 12 kb||600 ng||6.4 pmole||20.0 µL|
|L = 12 to 30 kb||ln(L) / 3
e.g. if L = 15 kb
ln(15)/3= 902 ng
|20 pmole||20.0 µL|
|L >30 kb||ln(L) / 2
e.g. if L = 33 kb
ln(33)/2= 1748 ng
|200 pmole||20.0 µL|
|BACs, Cosmids, etc.||2 µg
||200 pmole||20.0 µL|
Large templates can be difficult. Please discuss with the lab prior to submission.
|Size in base-pairs||Template amount (ng)||Primer amount (pmole)||Total Volume*|
|100-200||20 ng||6.4 pmole||20.0 µL|
|200-300||30 ng||6.4 pmole||20.0 µL|
|300-400||40 ng||6.4 pmole||20.0 µL|
|400-500||50 ng||6.4 pmole||20.0 µL|
(in bp) = X ng
|6.4 pmole||20.0 µL|
If you design your own primer, be sure to use data of known, high quality, i.e., phred-value >30.
Sample calculation: 20 µM primer stock contains 20 pmole/µL, so 0.32 µL contains 6.4 pmole.
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Fragment Analysis (E Mode)
- Perform labeled fragment generation for using your favorite protocol (see dye set info below).
- Clean up fragments to remove unincorporated nucleotides, primers, and salts.
- Final sample concentration must be at 100ng in 1uL; If multiplexing, samples must also be at 100ng in 1uL.
The maximum volume in each tube or well can ONLY BE 8uL (ex: If using G5, you can use the four dyes available twice, as long as the expected alleles vary in size. This would give a maximum value of 8uL. If you are unsure about how this works or would like to try other combinations please speak with us.)
We currently use:
Dye Set D-DS-30: 6-FAM (blue), HEX (green), NED (yellow), ROX (red)
Dye Set G5-DS-33: 6-FAM (blue), VIC (green)), NED (yellow), PET (red), LIZ (orange)
We use GS500 (-250) LIZ or ROX as the size standard.
Suggestions and Best Practices
If you are using Dye Set D you will not be able to label your samples with ROX.
If you are using Dye Set G5 you will not be able to label your samples with LIZ.
Use different dye labels for PCR reactions with overlapping product sizes.
Use a combination of dyes that display in different colors and can be detected by the same virtual filter set (dye set D or G5).
Use greater dye concentrations in PCR reactions with dyes of low emission intensity than for dyes of high emission intensity.
Do not multiplex primers with similar product lengths labeled with similar dyes.
Before performing PCR, check for primer compatibility-avoid excess regions of complementarity among the primers, choose ones with similar melting temperatures, and then test for successful co-amplification.
After samples have been run on the ABI 3730xl we will email your results. Applied Biosystems provides a free program (PC only) for analysis of their data files. We recommend this. Peak Scanner is a very simple program and we can provide directions for use. GeneMapper is available from AB/Lifetech for more convenient fragment analysis.