| SNGE,NIMH,NIH
http://intramural.nimh.nih.gov/lcmr/snge/Protocols/BACProtocols/BACSeq.html
Courtesy Lisha Xu and Alice C. Young, Molecular and Cell Biology Research and Development, Life Technolgies, Inc., Rockville, MD 20849
16 µl BigDye Terminator Ready Reaction Mix
1 µg BAC DNA (determined from gel)
10 pmol primer
water to 40 µl
Heat tubes at 95C for 5 min., then perform 30 cycles of:
95°C x 30 sec
55°C x 10 sec
60°C x 4 min
Run BACs on a 377 and load the entire sample after clean up. If you use a 3700, the loading would be very different.
This IS the double recipe. Normally a reaction is only 20 µl.
For sequencing we use ABIs "BigDye Terminator Cycle Sequencing Ready Reaction Kits v2.0 with AmpliTaq DNA Polymerase", cat. no. 4314414, 100 reactions. Avoid repeated freeze-thaw of ready reaction mix. Signal decreases with each thawing. To get a protocol order cat. no 4303237 (they are free, but must be requested).
Check with the group running the sequencer. Some 3700 users now are using the new BigDye 3 system. This is not available for 377 yet (different dyes require different filters). Be sure they know exactly what type of kit you used so they run the right detection and analysis parameters.
For clean up we use Princeton Separations (732 431-3338) CentriSep Columns, cat. no. CS-901, 100 columns.
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