Guidelines for DNA Sample Submission for High-Throughput Sequencing    

We accept any form of sample format for High-throughput DNA sequencing projects. Please see below for details on how samples should be submitted:

PURIFIED DNA SAMPLE 1. We accept plasmid DNA, PCR DNA, purified or unpurified in both 96-well and 384-well plates. 2. Properly seal the plates to prevent evaporation, cross-contamination, and damage during shipment. 3. Freeze the plates at -80°C and ship with dry ice.  The amount of template and primer required in each reaction is shown below: Template DNA Size of DNA Amount (ng) Primer (pmol) Plasmid DNA up to 10 kb 100 10 PCR DNA 100-200 bp 5 10 PCR DNA 200-500 bp 10 10 PCR DNA 500 bp-1 kb 20 10 PCR DNA 1kb-2 kb 50 10 PCR DNA over 2 kb 100 10 Large DNA over 10 kb 500-1,000 20 Please send enough DNA to do a re-run in case the first reaction fails.  Pre-mixed instead of sending in separate tube: Template DNA Size of DNA Amount (ng) Total Vol(µl) Primer (pmol) Plasmid DNA up to 10 kb 100 10 20 PCR DNA 100-200 bp 5 10 20 PCR DNA 200-500 bp 10 10 20 PCR DNA 500 bp-1 kb 20 10 20 PCR DNA 1kb-2 kb 50 10 20 PCR DNA over 2 kb 100 10 20 Large DNA over 10 kb 500-1,000 20 40 Note that if a template requires to be analyzed in two directions, pre-mixed samples of the template with both forward and reverse primers should be submitted as separate reactions.		LIGATION MIXTURES FOR TRANSFORMATION 1. Ligation mixtures are processed by electroporation to transform into suitable host cells. They are further propagated for sequencing or other molecular biology procedure. 2. Ligation mixtures should be ethanol precipitated, frozen in water, and shipped in a screw top tubes in a frozen (dry ice) package.  TRANSFORMATION SAMPLES FOR PLATING AND PICKING 1. Transformation samples should be frozen and shipped in a screw top tubes in a dry ice package. 2. Suitable antibiotics and their concentration for clone selection: Antibiotic Final Concentration Chloramphenicol 12.5 g / ml Carbenicillin/Ampicillin 50 µg / ml Kanamycin 35 µg / ml Zeocin* 25 µg / ml 3. Templates which contain Zeocin-resistant gene should be grown in low salt LB media. 4. Submit enough vials of frozen, never thawed, material each time since the titer usually drops after freezing and thawing. 5. Submit at least twice the number of required colonies for each sample and provide titer information on the order form.
PURIFIED DNA SAMPLE Sample Type/Format Sample Requirements Comments/Additional Requirements Bacterial agar plates 25 cm x 25 cm tray 200ml LB agar 1,500 - 2,000 colonies/lawn are required (no more than 3,000/lawn) 96-well bacterial cultures 200 µl media 10% final glycerol concentration Culture grown in LB media and incubated at 37°C for 12 hours static growth. Templates contataining the Zeocin-resistant gene should be grown in low salt LB media 384-well bacterial cultures 90 µl media 10% final glycerol concentration Culture grown in LB media and incubated at 37°C for 12 hours static growth. Templates contataining the Zeocin-resistant gene should be grown in low salt LB media PCR Products 96 or 384-well full skirted plates Minimum volume: 20 µl Conc.: 15 - 25 ng/µl Samples should be normalized across the plate Ligation mixture - for transformation At least 10 µl per sample send in frozen stock Note when the sample has been ethanol precipitated Transformation - glycerol stock for agar plating 200 µl aliquots 2X the number of requested clones should be submitted Primers Minimum of 5 nanomoles for 384 wells reads of fewer Primers should be ~20 bp in length, with Tm between 56°C and 62°C. Primers should be resuspended in dH2O (not TE buffer) Primers - plate format for sequencing of PCR priducts 25 µl of 3 µM primer/well Primers should be plated in the primer plates according to clone layout