Sanger sequencing standard Big Dye reaction

For all reactions performed at the facility we use a standard reaction mix and PCR protocol described below using the BigDye v3.1 Terminator Cycle Sequencing Kit (Thermofisher P/N AB0384/240; BS034042). We would recommend a similar reaction for users wishing to perform their own Big Dye reaction.

Big Dye Master Mix

Big Dye Reaction Mix

PCR protocol

SEQ FAST

Step1: 1 min at 96oC

Step 2: 10 sec at 96oC

Step 3: 5 sec at 50oC

Step 4: 1 min at 60oC

Step 5: Go to Step 2 for 24 cycles

Step 6: Hold at 12oC

The BigDye reactions are column purified, dried in a vacuum concentrator, resuspended in HiDi and denatured before running on the 3730XL.

For large projects this protocol can be adjusted as required and for more difficult samples additives can be used to improve the effectiveness of the BigDye reaction.