DNA Sequencing (Plasmid and PCR)

Plasmid sample

Plasmid DNAs purified through most commercial kits are suitable for DNA sequencing. Please follow the protocol of commercial kits. Low copy number plasmids need to be further concentrated before sequencing. These plasmid can be concentrated by ETOH precipitation, centricon spin column, or our template amplification service (RCA).

PCR sample

PCR products need to be purified to eliminate primers and dNTP. You can either use commercial available kits or run gel electrophoresis to purify PCR products. Check quality and concentration of PCR products by gel electrophoresis using a DNA standard of a defined concentration. Please do not overexpose the film when taking a gel picture.

We highly recommend that our customers purify PCR products before sequencing. However, unpurified PCR products are also welcome.

Premixed

• For double-stranded plasmid DNA (<10kb), submit 0.6ug total DNA mixed with 8pmol of primer in 12ul volume.
• For single-stranded plasmid DNA (<10kb), submit 0.3ug total DNA mixed with 4pmol of primer in 12ul volume.
• For Cosmid/BAC-DNA, submit 2ug total DNA mixed with 8pmol of primer in 12ul volume.
• For purified (kit purified, gel purified or exonulease/SAP treated) PCR product, submit 20-50ng per 1000 bp mixed with 8pmol of primer in total 12ul volume. For example, submit a total 25ng of 500-bp PCR fragment mixed with 8pmol of primer in 12ul volume.

Unmixed

• For double-stranded plasmid DNA (<10kb), submit 0.6ug total DNA in 10ul volume.
• For single-stranded plasmid DNA (<10kb), submit 0.3ug total DNA in 10ul volume.
• For Cosmid/BAC-DNA, submit 2ug total DNA in 10ul volume.
• For unpurified (directly come from PCR machine) PCR product, submit 20-50ng per 1000 bp in total 10ul volume. For example, submit a total 25ng of 500-bp PCR fragment in 10ul volume.

Primer walking (unmixed please)

• Double-stranded DNA template: 2ug of plasmid DNA for 1 kb insert, and 1ug for each additional kb.
• Single-stranded DNA template: 1ug of single-stranded DNA for 1kb insert, and 0.5ug for each additional kb.
• Please resuspend template DNA or dilute primer in distilled water.

High throughput sequencing sample preparation

Sample submission for high throughput sequencing should be in premixed format as samples submitted for the single-pass sequencing service unless using one of our common primers. Samples should be submitted in 96-well V-shaped PCR plate, and arranged by columns (A1-H1, A2-H2, ...)

Bacteria sample preparation

Individual bacteria culture tube

Shake the inoculated bacteria in sterile bacteria culture tube for more than 12 hours. Transfer the bacteria culture into a sterile eppendorf tube. Cap tightly and label clearly on the tube.

Please use parafilm wrap the tube and submit to us.

Transformation plate

After plasmid DNA transformation, incubate the agar for more than 12 hours. Make sure the bacteria side facing down.

Please use parafilm wrap the plate and submit to us.

High throughput bacteria culture plate

You may inoculate bacteria on a square share agar plate as in 96-well plate manner. After single colony inoculation, incubate the plate for more than 12 hours. Clearly label A01, H01, A12 and H12 position.

Animal's tissue and genomic DNA preparation

Animal's tissue

Collect a small piece of animal's tissue into a sterile eppendorf tube (for example, the tip of a mouse tail). Submit the tissue sample to us as soon as possible.

Animal's genomic DNA

Alternatively, you can also use commercial kits to purify genomic DNA from animal's tissue or blood. Please follow protocol of the commercial kit.