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) 4μl Primer (from mermade) (200 pmoles of primer) 1μl Polyoxyethylenesorbitan monolaurate (1% v/v with sterile double distilled water)* 1μl Igepal CA-630 (1% v/v with sterile double distilled water)* 1μl BigDye V3.0 (un-diluted) 1μl Final volume 9μl 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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