PROTOCOL

1.Termination mixes—Prepare the termination mixes on ice. Mix 2∝l of Nucleotide Master Mix (either dGTP or dITP—see note below) and 0.5∝l of [α-33P]ddNTP (G, A, T, or C—one of each per sequence) to produce a

termination mix for each ddNTP. Label, fill and cap four tubes (‘G’, ‘A’, ‘T’, ‘C’) with 2.5∝l of each termination mix. It is more accurate and convenient

to prepare batches of termination mixes sufficient for all sequences to be performed, then dispense 2.5∝l from this batch to each vial for the termination reactions. It is recommended that these batches of termination mixes be made up routinely.

To prepare termination mixes for (n) reactions, mix:

 

G

A

T

C

Nucleotide Master Mix

(2 x n)∝l

(2 x n)∝l

(2 x n)∝l

(2 x n)∝l

[α-33P]ddNTP

(0.5 x n)∝l

(0.5 x n)∝l

(0.5 x n)∝l

(0.5 x n)∝l

 

––––––––– ––––––––– ––––––––– –––––––––

Total

(2.5 x n)∝l

(2.5 x n)∝l

(2.5 x n)∝l

(2.5 x n)∝l

Note: The termination tubes can be left uncapped until all reagents have been added if the tubes are kept on ice and the reaction mixture is added within a few minutes. For determination of new sequences, or of sequences with high G-C content, the dITP Nucleotide Master Mix is recommended. This will eliminate all compression artifacts but will result in somewhat uneven band intensities, especially in the ‘G’ lane. When perfectly uniform band intensities are desired, such as when examining sequences from potentially heterozygous individuals, the dGTP Nucleotide Master Mix should be used.

2.Reaction mixture:

For multiple (n) reactions with different primers and/or templates, prepare a n+1 batch of reaction buffer, water, polymerase and aliquot; then add the unique primer and/or template in the appropriate concentration and volume to the aliquots.

Reaction Buffer

2∝l

 

DNA

_∝l* (50-500ng or 25-250fmol)

Primer

_∝l* (0.5-2.5pmol)

H2O

_∝l

(To adjust total volume to 20∝l)

Thermo Sequenase polymerase (4U/∝l) 2∝l

(8 units polymerase—add LAST)

Total

–––

 

20∝l

 

*For the control reaction, use 10∝l of control DNA and 1∝l of control primer.

9