Sequetech has developed a sequencing chemistry (BDX chemistry) which is effective on a wide range of problematic templates. Such templates may arise during the course of genomic sequence finishing and gap closure, shRNA and siRNA cloning in RNAi vectors, Ribosomal RNA gene sequencing, long terminal repeat sequencing (LTR), analysis of repetitive sequences, etc.
Hard stops, where the signal exhibits a large and abrupt drop in intensity (Fig. 1), are caused by secondary structures such as hairpins, stem/loops or triple helices.
These structures result from certain sequence motifs including inverted repeats, palindromes, etc. Also, high GC content templates frequently exhibit hard stops or a noisy, weak sequencing signal.
(Fig. 1) A sample sequenced with regular Sanger Sequencing reaches an abrupt stop at bp 210
(Fig. 2) Our proprietary BDX chemistry is effective at getting through both hard stops and high GC content sequences.
BDX Chemistry tends to work best with plasmids. If you are working with PCR products, Sequetech has developed a propriety sequencing protocol called Turbo PCR. Please contact us to determine the best workflow for your samples
BDX Chemistry was specifically formulated for difficult templates but has the same sample requirements as our standard sequencing chemistry:
Template Requirements (BDX)
| Template Type | Amount | Concentration | Notes |
|---|---|---|---|
| Plasmids * | 200ng/Primer Extension | >50ng/ul | Submitting extra will allow for re-runs, optimization and troubleshooting. Primers and templates are stored for at least 3 months. We strongly recomment confirming PCR products by gel analysis. |
*Turbo PCR Sequencing is recommended for sequencing problematic PCR products.
| Amount | Minimum Concentration |
Notes |
|---|---|---|
| 4 pmole/primer extension | 2 uM | Submitting extra will allow for optimization and troubleshooting. Primers and templates are stored for at least 3 months. |
Have any questions about the service or would like pricing? Please email us at info@sequetech.com or call 650-967-8685.
