Standard Service- PCR:
Send purified PCR products pre-mixed with a custom primer. Products are then cycle sequenced using Big Dye Terminators. After a purification step to remove unincorporated dye terminators products are analyzed on the 3730xl.
Preparation of PCR Product for Submission
10ng of PCR product per 100nt
1μL of 5μM (micromolar) primer (NOTE: 5 μM is equal to 5 pmol/μL)
Bring to a total volume of 10 μL with molecular grade water (see here shipping instructions)
The amount of PCR product to use depends greatly on length and purity. Our recommendations are a starting point, more or less may be required depending on the template and the method of estimating the concentration. A nanodrop is the best way to estimate concentration, but an agarose gel should be used to look at products to confirm there is a single amplicon. When estimating concentration make sure to analyze the same products being sent to us-don’t perform a gel purification and assume 100% recovery.
Standard Service- Plasmid:
Send purified plasmids pre-mixed with primer. Products are then cycle sequenced using Big Dye Terminators. After a purification step to remove unincorporated dye terminators products are analyzed on the 3730xl.
Start with between 500ng-1000ng of plasmid DNA for <6 kb plasmids (more or less may be required depending on the total length of Plasmid). If using a Nanodrop for quantification the 260/280 ratio should be ~1.9, higher is fine, lower is an indication of contamination.
Add 1μL of 5μM (micromolar) primer (NOTE: 5 μM is equal to 5 pmol/μL)
Bring to a total volume of 10 μL (see here for tube/plate labeling and packaging instructions)
Standard Service- Difficult Templates:
For difficult to sequence templates we suggest requesting Enhancer Solution to be added to your samples. This is an additional cost but has improved the quality of reads for GC rich templates, shRNA and hairpins.
Standard Service- BAC/Fosmid:
For this service you’ll be sending purified Bacterial Artificial Chromosomes or Fosmid templates pre-mixed with primer. Products are then cycle sequenced (using a modified protocol) and Big Dye Terminator at 1/2 Chemistry. After a final purification step to remove unincorporated dye terminators your products are analyzed on the 3730xl. Orders are submitted and data is posted on GeneSifter, our lab information management system.
Purification of BAC’s
Our users recommend either the Qiagen Large Construct Kit for purification of up to 50 μg BAC or the Qiagen DNEasy Blood and Tissue kit
At the end of the Blood and Tissue kit procedure, there will be 2 aliquots of 200 μl each in Qiagen’s ”AE” elution buffer. Precipitate this with 100% EtOH, wash 2x w/ 70% EtOH, and resuspend in a small volume of 10 mM Tris, pH 8.
Purification of Fosmids
Our users recommend the Epicentre FosmidMAX DNA Purification Kit
Follow Manufacturer’s Instructions. Always inoculate with fresh clones.
Our users report success with determining concentration using a Nanodrop. A260/A280 ratios for BAC and Fosmid templates are usually in the 1.8-1.9 range, or one can use a Qubit with the appropriate kit. We have a Qubit available for use, contact us for more information.
For BAC’s send 2-6 μg of template. Sometimes the yield from DNA purification isn’t sufficient to allow this, so less template down to about 2 μg could produce some results.
For Fosmids send .5-1.5 μg. It may be best to try multiple/varying concentrations to find what is the ideal concentration based on template quality.
To your template add 10 pmol primer
Bring to a total volume of 11μl (see here for tube/plate labeling and packaging instructions)
Select Standard Sequencing with these changes to the GeneSifter order form:
Choose Plasmid DNA Type, but under purification method note that this is a BAC or Fosmid.
Choose Alternative Chemistry: 1/2 Chemistry
Include the following instructions in the Order Comment field:
BAC or Fosmid Template:
Add 4uL of Big Dye
With cycling parameters
95degreesC for 5 minutes
95degreesC for 30 seconds
55degreesC for 20 seconds
60degreesC for 4 minutes
Run Ready with Purification
Send unpurified cycle-sequenced samples (with ice packs or dry ice in warmer months). Samples with volumes lower than 5μl will have water added. Samples will be purified and run at the next available time slot. We can run products sequenced with Big Dye Terminator v3.1 from ThermoFisher Scientific. (If you need v1.1, contact us).