FGB 2/2002

ABI Prism dRhodamine Terminator Cycle Sequencing Ready Reaction Kit

C-346, 1-1812 http://dir.niehs.nih.gov/dirlmc/seqcore.htm

Sequencing setup:

DNA (0.5*-3ug) X

Oligo (M13 For or Rev usually) 0.1*

DRhodamine or BD: 8

H20 up to 20ul

*Recommended for pDNA. Use 50-100ng for single stranded DNA and 30-90ng for PCR products.

*Use 0.1ul of 100ng/ul oligo’s (M13 For, Rev) and for 10uM stocks. Or dilute 1:10.

Use 1ul of sequencing oligo’s, which are 10ng/ul. 3.2 pmol is recommended.

Sequencing reaction without oil (faster cycles):

Stage 1, step 1: 96’C for 2 minutes 1 cycle

Stage 2, step 1: 96’C for 10 seconds 25 cycles

Stage 2, step 2: 50’C for 5 seconds

Stage 2, step 3: 60’C for 4 minutes

Hold at 4’C

My 10uM oligo stocks are about 50-100ng/ul. My sequencing oligo’s are diluted 1:10 from 100ng/ul thus are 10ng/ul. For PCR and microarray, they are not diluted so only use 0.5ul. You only need about 4pmol of oligo. Use BD for high G-C rich sequences.

Dye clean up. Use Qiagen DyeEx kit for either preperation.

For dRhodamine (not BD) you can precipitate with 50ul of 100% ETOH + 2ul 3M sodium acetate (pH4-5), place at –80’C for 30min and then spin for 15min at 4’C. Wash with 250ul 70% ETOH in a 1.5ml tube. Spin again and then dry down 5-10min and it is ready to go! Do not re-dissolve.