| Protocol for Sequencing Very Difficult Regions
Ziyun Yao and B.A. Roe
02-14-2002
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.
NOTE:
* 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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