Protocol for Sequencing Very Difficult Regions

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~undefined  1μl

Igepal CA-630 (1% v/v with sterile double distilled water~undefined                                   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: