Taq-polymerase catalyzed cycle sequencing using fluorescent-

Thursday, November 20, 2003

Description
Each base-specific fluorescent-labeled cycle sequencing reaction routinely included approximately 100 or 200 ng Biomek isolated single-stranded DNA for A and C or G and T reactions, respectively. Double-stranded cycle sequencing reactions similarly contained approximately 200 or 400 ng of plasmid DNA, isolated using either the standard alkaline lysis or the diatomaceous earth modified alkaline lysis procedures.

Procedure
1. Pipette 1 or 2 ul of each DNA sample (100 ng/ul for M13 templates and 200 ng/ul for pUC templates) into the bottom of the 0.2 ml thin-walled reaction tubes (Robbins Scientific). Use the 1 ul sample for A and C reactions, and the 2 ul sample for G and T reactions. Meanwhile, preheat the PE Cetus Thermocycler 9600 to 95degC (Program #2).

2. Prepare the Taq polymerase dilution. AmpliTaq polymerase (N801-0060) is from Perkin-Elmer Cetus.

30 ul AmpliTaq (5U/ul)
30 ul 5X Taq reaction buffer
130 ul ddH20
190 ul diluted Taq for 24 clones

3. Prepare the A, C, G, and T base specific mixes by adding base-specific primer and diluted Taq to each of the base specific nucleotide/buffer premixes:
A,C/G,T
60/120 ul 5X Taq cycle sequencing mix
30/60 ul diluted Taq polymerase
30/60 ul respective fluorescent end-labeled primer
120/240 ul

4. Seal the reaction tubes carefully with the strip caps, and centrifuge briefly at 2500 rpm. Place the tube/retainer set in the 9600 Cycler, abort the soak file program, and run program #11. This program will cycle the sequencing reactions for 30 cycles of seven temperatures (30 cycles of 95degC denaturation for 4 seconds; 55degC annealing for 10 seconds; 72degC extension for 1 minute; 95degC denaturation for 4 seconds; 72degC extension for 1 minute; 95degC denaturation for 4 seconds; and 72degC extension for 1 minute), and then will link to a 4degC soak file until that program is aborted. (It is possible to freeze the reactions at -20degC after cycling, prior to the pooling step).
5. Briefly centrifuge the plate to reclaim condensation. Pool the four base specific reactions into 250 ul of 95% ethanol.

6. Precipitate the sequencing reactions, and store the dried samples at -20deg C.

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Taq-polymerase catalyzed cycle sequencing using fluorescent-