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Hybridization of primer to template and etha nol precipitation Add, to each tube:
Primer 3.5 10 ng conc. for 15 mer oligo
H20 3.5
3M NaOAc 3
(pH 4.5)
Mix. Then add:
Etch 75
(ice cold)
Dry ice x 10 min. Spin 15 min at 40C. Wash with 70% ethanol. Dry in speedvac. Meanwhile, label tubes for termination step and add appropriate amount of TM to each. We use Nuncolon 72 well microplates and place on 370C heating block.
Labeling Reaction
Add to each pellet:
H20 8
5x Buffer 2
(Sequenase)
5 min at R.T., then:
.lM DTT 1
Diluted LM 2 (Labeling Mix GII: dilute 1:15 to sequence
close to primer; 1:5 to sequence further
35SdATp away.)
Diluted .5 (1000-1500Ci/mM; l0mCi/ml)
Sequenase 2 (Dilute 1:8 in dilution buffer)
5 min at 370C
Terminations
TM'S:GATC 2.5 (Appropriate Termination Mix into each tube)
370C x 1 min
LR 3.5 (Aliquots of the above Labeling Reaction)
5 min at 370C
stop
To each tube add:
Stop soln 4
Store at -200C until ready to load gel
0
USB (cont'd)
Prepare samples for gel
800C x 2 min before loading
Most people use 6% acrylamide
50% urea (w/v)
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