Protocol for Sequencing Very Difficult Regions

Ziyun Yao and B.A. Roe



Target DNA (SAP-ExoI cleaned PCR 7-deaza-dG containing product)                                 4ul

Primer (from mermade) (200 pmoles of primer)                                                                     1ul

Polyoxyethylenesorbitan monolaurate (1% v/v with sterile double distilled water)*             1ul

Igepal CA-630 (1% v/v with sterile double distilled water)*                                                  1ul

BigDye V3.0 (un-diluted)                                                                                                        1ul

Final volume                                                                                                                            9ul


Sequencing reaction conditions:

95 degree C for 5 minutes, followed by:

99 or 60 cycles for sequencing on the 377 (or BaseStation) or 3700, respectively, of:

95 degrees C for 30 seconds

50 degrees C for 20 seconds

60 degrees C for 4 minutes

4 degrees C hold

Filter through Sephadex columns in 96 well microtiter plate format for loading on either the ABI 377 or the MJ BaseStation, or ethanol precipitation for loading on the ABI 3700.



* Polyoxyethylenesorbitan monolaurate (1% v/v Tween 20, Sigma-Aldrich # P-9416 with sterile double distilled water), Igepal CA-630 (1% v/v Octylphenoxy)polyethoxyethanol [which is identical to NonidetP-40, a nonionic detergent], Sigma-Aldrich # I-8896 with sterile double distilled water) (both detergents are added to a final concentration of 0.1%), proline (Sigma-Aldrich # P-5607) and/or MMNO (4-Methylmorpholine N-oxide) (Sigma-Aldrich # 22428-6) (where proline and MMNO are at a final concentration of from 0.4M to 0.8M) may facilitate reading through G/C rich regions both in PCR and sequencing reactions.  See Life Technologies European Patent Number WO09946400.


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Bruce Roe,