| Recommended concentration and volume |
| Sample type |
Concentration |
Minimum volume |
| E.Coli. Bacterial Culture |
200-300 μl E.Coli. (fresh liquid culture > 8 Hours) in 1.5ml tube |
| monoclonal agar plate ( culture > 8 Hours), numerically label each colony on the plate. |
| Plasmid |
< 5 Kb |
> 50 ng/μL |
20 μl (enough for at least 6 reactions) + 5 μl per add’1 reaction |
| 5-10 Kb |
> 100 ng/μL |
| > 10 Kb |
> 200 ng/μL |
| Purified PCR product |
< 500 bp |
5 ng/μL |
| 500 – 1500 bp |
15 ng/μL |
| > 1500 bp |
30 ng/μL |
| Unpurified PCR product |
Same requirement as Purified PCR product (see above) |
30 μl (enough for at least 6 reactions) + 5 μl per add’1 reaction |
| Primer |
5 pmol/μl |
10 μl (enough for at least 6 reactions) + 2 μl per add’1 reaction |
| Premixed with primer |
Template and primer should follow the concentrations listed in the above prior to mixing |
6μL Template + 6μL Primer |
| Ready to load Plate |
Total reaction volume 5-20μl |
| Sample type |
| • BAC: Plasmid will be extracted for sequencing. |
| • Plasmid: Template Quantitation by gel will be done before sequencing. |
| • Unpurified PCR: Magnetic Beads Purification which could purify the single-band PCR product, will be done. |
• Purified PCR: while sample had been cleaned up with ExoSAP-IT Reagent, CleanSweepTM PCR Purification Reagent, or Spin Columns, and so on.
If the target size of PCR products is not more than 500 bp, please fill the accurate length.
We will double check the concentration records accordingly. |
| • Premixed: Primer is pre-mixed with sample, submit in a 0.5 ml EP Tube, please mark “Pre-mixed” on the sample bag for quick sequencing service. |
| • Ready to load plate |
| ···· Only available for plates. |
| ···· Only ABI BigDye Terminator Kit (v3.1) will be accepted in cycle sequencing. |
| ···· Considering the potential dye degradation in long time shipment, dried samples with dye terminator purified are preferred. |
| ···· Available purify service if need. |
| ···· In long time shipment, preserve samples with dry ice is highly recommended. |
| DNA preparation |
| • All the DNA submitted should be in deionized water only. |
| • Quantitation of template by gel is highly recommended. |
| • For plasmid sequencing, run the colony PCR before sequencing is recommended. |
| Sample submission |
| • Please sparate the order according to the sample tpye, and enclose a copy of order information with every shipment. |
| • Please make sure the DNA samples are securely packaged to prevent sample damage during delivery. |
| • The dropbox sample collection will be done before 10 a.m on working day. Please contact us for the schedule. |
| Tube |
| • For 15 or fewer samples, please use 0.5ml safe-lock tubes (e.g. Eppendorf) at room temperature. |
| • For your convenient, you could mark the necessary information as the sample name, in the sequencing order form. |
| • But, keep sample names simple with a maximum of 25 characters long (only English alphabet and digit). And ensure that samples are clearly labeled. |
| • To make the tube marker brief and clear, we suggest to write just the code ( like Sample No.1, 2, 3, and so on ) on the tube. |
| Plate |
|
|
|
| • For 16 or more samples, please submit them in 8 strip 0.2ml tubes or 96-well plates. |
| • Indicate your order number and plate name on the plate frame or enclose printed order sheet with samples for our reference. |
| Primer type |
| • Universal primer: 368 free primers would be provided for sequencing.(see the ‘Universal Primer List’ sheet) |
| • Enclosed primer: should be provided in deionized water,submit in a 1.5 ml EP Tube, attached with a paper order form. |
| • Synthesis primer: primer sequence filled in the order form will be synthesized accordingly. |
| • Kept primer: primer code could be found in your history order. |
| Primer preparation |
| • BGI provides more than 100 universal primers for free. (see the ‘Universal Primer List’ sheet) |
| • Tips for designing primer |
| ···· A melting temperature (Tm) between 40-60°C |
| ···· GC content is 40-60% |
| ···· The length of the primer should be 18-25 bases. |
| ···· It is recommended that primer binding site should be 50-100 bases before the start of your sequence of interest. This is because the first 30-50 based of sequence are usually messy and unreliable. |
| ···· High Purity |
